-NRLF 


Dr. 

Gift   of 
James     Hopper,   Jr. 

HYDRATION  AND  GROWTH 


BY 
D.  T.  MACDOUGAL,  PH.  D.,  LL.  D. 

Director  of  the  Department  of  Botanical  Research 
Carnegie  Institution  of  Washington 


PUBLISHED  BY  THE  CARNEGIE  INSTITUTION  OF  WASHINGTON 
WASHINGTON,  1920 


CARNEGIE  INSTITUTION  OF  WASHINGTON 
PUBLICATION  No.  297 


PRESS  OF   GIBSON  BROTHERS,   INC. 
WASHINGTON,  D.  C. 


I  \ 


PREFACE. 

Three  main  conceptions  concerning  growth  and  its  developmental 
aspects  in  plants  are  to  be  met  in  the  history  of  physiology  in  the  half 
century  beginning  with  the  researches  of  Sachs  and  his  school.  The 
first  or  earliest,  that  of  special  stuffs  or  substances  necessary  for  the 
initiation  of  growth  and  differentiation  of  various  organs,  especially 
for  the  origination  and  development  of  reproductive  organs,  is  now 
giving  way  to  the  modern  conclusion  that  "formative"  material  as 
such  has  no  actual  existence  in  fact  and  no  good  basis  in  theory.  The 
present  trend  of  thought  leads  to  the  assumption  that  growth 
proceeds  from  and  depends  upon  states  or  combinations  of  material 
or  accumulations  in  connection  with  living  matter  rather  than  upon 
any  special  constructive  stuff  or  substance.  This  may  also  be  held 
to  apply  to  hormones,  vitamines,  and  other  symbolic  expressions  for 
combinations  of  material  necessary  for  initiating  and  maintaining  de- 
velopment, reproduction,  or  growth. 

The  second  aspect  of  the  subject  is  that  which  deals  with  the  incor- 
poration of  new  material  in  the  cell  and  its  subsequent  distention  by 
an  osmotic  mechanism,  upon  the  basis  of  researches  of  Pfeffer  and 
de  Vries.  The  protoplast  is  dealt  with  as  a  sac.  The  products  of  the 
metabolic  processes  converge  in  the  vacuole,  which  in  consequence 
becomes  the  seat  of  osmotic  forces  and  the  center  of  the  mechanism  of 
distention.  An  important  feature  of  this  scheme  of  operation  is  an 
ideal  "semi-permeable"  membrane,  not  morphologically  identifiable, 
internal  to  the  cellulose  wall  or  other  durable  and  visible  integument. 
The  exploitation  of  the  theory  of  permeability  of  this  membrane  has 
been  carried  out  in  such  manner  as  to  place  undue  emphasis  on  the 
action  of  the  external  layer  of  the  protoplasm.  The  basic  conception 
of  the  diffusion  of  material  into  the  vacuole  remains  sound,  and  the 
differentiating  action  of  protoplasm  by  which  the  increase  in  the  con- 
tents of  the  vacuole  sets  up  an  internal  pressure  expressed  as  turgidity 
is  undeniable.  But  the  attempt  to  base  all  features  of  water-relations, 
turgidity,  and  growth  upon  the  action  of  solutions  has  been  proved 
inadequate  and  has  resulted  in  an  obvious  neglect  of  the  play  of  molec- 
ular forces  in  surface  tensions,  in  imbibition,  and  other  activities  of 
matter  in  a  colloidal  state. 

The  third  group  of  inquiries  has  been  directed  toward  measurement 
for  the  purpose  of  establishing  the  physical  constants  of  growth. 
Auxesis  or  developmental  enlargement  in  living  things  has  been  mis- 
takenly dealt  with  as  a  unified  process,  or  as  a  series  of  successive 
reactions  in  studies  of  temperature  effects,  by  many  writers,  and 
coefficients  of  some  apparent  validity  within  a  small  part  of  the  range 
within  which  growth  takes  place  have  been  found.  Growth  is  a  con- 
stellation of  activities  and  the  rate  of  one  of  these  dependent  on  tem- 
perature may  be  the  determining  one  when  the  particular  process  forms 
either  the  retarding  or  leading  agency.  At  other  times  the  relative 


rv  Preface. 

rates  of  metabolism,  respiration,  hydration,  and  diffusion  may  coincide 
in  such  manner  as  to  make  possible  the  application  of  a  simple  formula 
for  the  effects  within  a  range  of  15°  or  16°  C.  The  relation  of  growth 
to  temperature  for  any  plant  between  10°  and  50°  C.  is  not  to  be 
expressed  by  any  simple  formula.  The  same  general  statement  may 
be  made  concerning  light  and  other  agencies,  none  of  which  has  received 
more  than  a  fraction  of  the  amount  of  attention  which  has  been  paid 
to  temperature  effects. 

The  assumption  as  to  the  general  identity  of  protoplasm  in  plants 
and  animals,  or  even  in  plants  as  a  group,  is  one  which  operates  to  stifle 
analytical  investigations  in  a  subject  of  this  kind.  The  relative 
amounts  of  proteins,  carbohydrates,  lipins,  and  salts  in  the  two  groups 
differ  widely.  In  addition  to  the  capacity  of  the  plant  to  synthesize 
carbohydrates,  amino-acids,  etc.,  which  the  animal  can  not,  the  res- 
piration and  metabolism  of  the  plant  are  predominantly  carbohydrate, 
while  those  of  the  animal  are  proteinaceous  to  a  much  larger  extent. 
It  would  seem  obvious  that  a  protoplasm  rich  in  fats,  high  in  proteins, 
and  permeated  with  their  derivatives  would  display  an  imbibition  and 
growth  different  from  living  matter  in  which  the  base  is  chiefly  the 
comparatively  physiologically  inert  pentosan  groups  and  which  neces- 
sarily adsorb  the  salts  and  acids  in  a  characteristic  manner.  The 
unities  or  general  properties  of  the  protoplasm  of  widely  different 
organisms  do  not  rest  upon  the  presence  or  proportion  of  elements  or 
compounds  so  much  as  upon  the  manner  in  which  the  necessary  con- 
stituents are  brought  together.  This  indispensable  condition  of  life 
is  the  colloidal  state,  in  which  the  substances  of  living  matter,  form  a 
semi-solid  or  elastic  gel  consisting  of  over  90  parts  water.  The  mole- 
cules are  large,  slow-moving,  and  adhere  to  form  aggregates  as  con- 
trasted with  the  separation  of  molecules  in  the  water  of  solutions.  This 
colloidal  structure  may  be  profitably  likened  to  that  of  a  house  or  fac- 
tory, serving  simply  as  the  scene  of  metabolic  processes  which  take 
place  under  special  conditions  of  surface  tension.  The  colloidal  labora- 
tory may  be  in  the  form  of  an  emulsion,  a  reticulum,  a  sponge,  a  crystal- 
line or  lamellar  structure,  with  corresponding  effects  on  metabolism, 
while  the  products  of  respiration  may  in  turn  cause  alterations  in  the 
chambers  in  which  it  takes  place. 

The  purpose  of  the  present  work  has  been  to  study  growth  upon  the 
basis  of  a  more  inclusive  conception  than  that  usually  implied  in 
osmosis.  The  total  absorbing  capacity  of  a  cell  or  mass  of  protoplasm 
for  water  is  regarded  as  being  exercised  in  the  process  of  hydration. 
The  source  of  energy  in  growth  and  swelling  is  the  unsatisfied  attrac- 
tion of  molecules,  or  particles  or  ions  bearing  an  electrical  charge. 
Substances  made  up  in  this  manner  may  unite  with  definite  propor- 
tions of  water  which  becomes  part  of  a  symmetrical  chemical  structure, 
the  union  being  known  in  classical  chemistry  as  hydration.  In  addi- 
tion, however,  it  is  known  that  such  particles  may  also  adsorb  and  hold 
in  combination  additional  molecules  of  water,  an  action  especially 


Preface.  v 

characteristic  of  swelling  in  colloids,  and  the  term  hydration  is  used  in 
the  present  work  to  include  the  entire  range  of  action. 

The  method  of  study  employed  has  been  one  in  which  biocolloids 
have  been  compounded  from  pentosans  and  proteins  in  proportions 
simulating  those  of  the  plant,  and  the  total  range  of  swelling  of  thin 
plates  of  this  material  has  been  measured  by  the  auxograph  which  has 
been  developed  for  this  purpose.  Series  of  measurements  of  such 
material  have  been  arranged  to  run  parallel  with  measurements  of  the 
unsatisfied  hydration  capacity  of  living  cell-masses  and  of  dehydrated 
tissues. 

The  acids  and  salts  have  been  employed  in  concentrations  mostly 
within  the  range  of  the  biological  possibilities.  It  follows,  therefore, 
that  these  substances  have  been  applied  in  solutions  in  which  complete 
or  nearly  complete  dissociation  has  taken  place. 

It  was  deemed  of  the  greatest  importance  to  traverse  a  wide  field  of 
possibilities,  which  made  the  use  of  simple  methods  advisable,  and 
solutions  have  therefore  been  applied  in  terms  of  molar  or  normal  con- 
centration, and  acidity  has  been  determined  by  titrations.  The  im- 
portance of  determinations  of  the  acidity,  especially  of  the  cell-sap, 
and  its  expression  in  terms  of  hydrogen-  or  hydroxyl-ion  concentration 
would  be  greater  in  any  more  critical  study  of  the  features  of  colloidal 
and  protoplasmic  action  discussed,  although  it  is  not  to  be  taken  for 
granted  that  this  is  the  dominant  or  determining  factor  in  all  cases. 

The  use  of  simple  methods  has  served  to  reveal  the  general  hydra- 
tion relations  of  plant  protoplasm,  the  influence  of  acidity  and  tem- 
perature upon  growth  and  swelling,  and  to  uncover  the  special  effects 
of  the  amido-compounds  upon  hydration  and  their  suggested  possi- 
bilities in  affecting  growth. 

The  significant  water-relations  of  the  cell-colloids  are  not  entirely 
included  in  direct  reactions  of  the  kind  mentioned,  however.  As  will 
be  described  in  Chapter  VII,  the  exigencies  of  plant  life  include  condi- 
tions under  which  dehydration  of  the  plasmatic  colloids  may  reach 
such  a  degree  that  the  nature  of  some  of  the  sugars  in  growing  cells 
may  be  affected,  and  one  of  these  changes  is  the  conversion  of  poly- 
saccharids  with  a  low  hydration  coefficient  to  pentosans  with  a  high 
hydration  capacity,  with  the  resulting  succulency  or  xerophily  of  the 
tissues  in  which  this  takes  place. 

I  am  indebted  to  my  colleagues  for  suggestions  and  assistance  both 
in  the  experimentation  and  in  the  preparation  of  the  manuscript, 
especially  to  Dr.  H.  A.  Spoehr,  who  has  collaborated  in  previous  papers 
and  who  has  given  continued  cooperation  and  valuable  advice  on 
various  phases  of  the  work  presented  here. 

DESERT  LABORATORY,  D.  T.  MACDOUGAL. 

Tucson,  Arizona,  1919. 


CONTENTS. 

PAGE. 

1.  Growth  and  colloidal  reactions 1 

II.  Fundamental  features  of  phytocolloids 11 

III.  The  constituents  of  biocolloids  which  affect  hydration  and  growth 27 

IV.  The  effect  of  salts  and  acids  on  biocolloids  and  cell-masses 37 

V.  The  effects  of  organic  acids  and  their  amino  compounds  on  hydration  and 

growth 54 

VI.  Reactions  of  biocolloids  and  cell-masses  to  culture  solutions,  bog,  swamp,  and 

ground  water,  and  other  solutions 65 

Vll.  Fluctuating  or  alternating  hydration  effects.     Basis  of  xerophily  and  succu- 
lence    78 

V11I.  Water  deficit,  or  unsatisfied  hydration  capacity 92 

IX.  Temperature  and  the  bydration  and  growth  of  colloids  and  of  cell-masses. ...  110 

X.  Imbibition  and  growth  of  Opuntia 128 

XI.  The  hydration  reactions  and  growth  of  Mesembryanthemum,  Helianthus,  and 

Phaseolus 145 

XII.  Water-content,  dry  weight,  and  other  general  considerations 161 

Literature  cited 174 

VI 


HYDBATION  AND  GROWTH. 


BY  D.  T.  MACDOUQAL. 


I.  GROWTH  AND  COLLOIDAL  REACTIONS. 

Growth  consists  in  increases  in  volume  of  masses  of  living  matter, 
usually  but  not  invariably  accompanied  by  accretions  of  material 
other  than  water  to  the  colloids  of  the  protoplasm.  Auxetic  changes 
in  members,  organs,  or  cells  of  the  larger  plants  may  be  readily  deter- 
mined by  external  measurements,  and  the  greater  part  of  the  available 
information  concerning  the  subject  has  been  obtained  in  this  manner. 
Many  generalizations,  however,  rest  upon  data  secured  by  taking  the 
gross  weight  of  organisms;  in  other  cases  the  dry  weight  is  used  as  a 
criterion,  a  method  which  obviously  may  be  used  only  in  securing  end 
or  total  results.  A  count  of  the  number  of  individuals  may  afford  a 
reliable  basis  for  the  estimation  of  the  rate  of  growth  and  multiplica- 
tion of  unicellular  organisms  such  as  bacteria,  in  which  the  limits  of 
enlargement  of  the  individual  are  quickly  reached.  Much  of  the  value 
of  the  results  presented  in  the  present  volume  is  to  be  attributed  to 
methods  by  which  the  varying  dimensions  of  organs  and  of  individual 
plants  were  followed  not  only  through  the  entire  period  of  develop- 
ment, but  alterations  accompanying  maturity  were  measured  with 
some  precision.  The  information  thus  secured  made  it  possible  to 
interpret  the  effects  of  the  ever-changing  daily  complex  of  environic  fac- 
tors and  to  evaluate  to  some  extent  the  effects  of  previous  experience 
upon  the  behavior  of  a  growing  organ  at  any  stage  of  its  development. 

Thus,  for  example,  the  action  of  a  cell-mass  at  any  given  tempera- 
ture is  influenced  not  only  by  the  degree  of  the  temperature  and  other 
environic  conditions  at  that  tune,  but  also  by  the  previous  experience 
with  these  factors,  particularly  temperature.  This  "  memory ' '  of  ante- 
cedent impressions  is  not  psychological  in  any  sense,  but  rests  upon 
definite  properties  of  colloids  which  are  known  or  are  measurable. 

The  diversity  of  constitution  and  consistency  and  variation  in  col- 
loidal condition  of  living  matter  is  so  great  as  to  evade  exact  or  detailed 
description.  But  the  general  composition  of  protoplasm,  the  character 
of  its  activities,  the  mode  or  manner  of  changes  in  its  colloidal  states, 
and  a  measure  of  the  factors  affecting  its  activities  may  be  compre- 
hended without  leaning  upon  vitalistic  conceptions  or  resorting  to 
mysticism  in  any  form.  The  fundamental  and  ultimate  structure  or 
architecture  of  protoplasm  is  a  result  of  the  force  of  surface  tension  and 
is  a  gel  in  which  the  solid  material  occurs  in  two  main  states  or  phases 


2  Hydration  and  Growth. 

with  water.  In  the  more  liquid  phase  the  molecules  of  the  substance 
are  associated  with  such  a  large  proportion  of  water  as  to  be  in  a  sus- 
pended condition,  while  in  the  more  solid  phase  the  proportion  of 
water  is  much  less.  This  phase  has  a  distinct  architecture  which  has 
been  likened  to  that  of  a  mesh,  felt,  foam,  or  honeycomb,  in  which  the 
denser  phase  forms  the  framework  and  the  fluid  fills  the  interstices. 
Under  certain  conditions  the  phases  may  be  reversed,  and  the  solid 
particles  be  rounded  into  globules  entirely  surrounded  by  the  fluid. 
These  structures  are  far  too  minute  to  be  visible  under  the  microscope, 
as  the  particles  which  are  dispersed  in  the  liquid  or  are  aggregated  in  the 
denser  structure  ma  each  consist  of  a  few  molecules  only.  In  addi- 
tion to  the  material  in  the  actual  sponge  of  protoplasm,  some  of  the 
same  or  other  substances  may  be  present  in  dispersions  or  solutions  in 
cavities  and  spaces  in  the  cell,  which  result  from  morphological  or 
mechanical  action  of  the  protoplast  (see  p.  21). 

The  aggregation  of  molecules  of  a  substance  in  a  colloidal  condition 
such  as  that  noted  above  is  a  more  complex  matter  than  that  of  the 
solution  of  a  crystalloidal  compound,  as,  in  addition  to  the  forces  of 
chemical  combination,  surface  tension  results  in  adsorption  or  union 
of  substances  in  indefinite  proportions. 

Four  main  groups  of  substances  make  up  the  protoplastic  engine — 
carbohydrates,  proteins  and  their  derivatives,  the  lipins,  and  the  salts. 
Perhaps  all  carbohydrates  may  exist  in  a  colloidal  condition,  but  the 
group  polysaccharids,  including  the  pentosans,  are  the  most  important 
in  the  architecture  of  the  protoplasmic  mesh,  as  these  substances  with 
proteinaceous  compounds  appear  to  determine  the  water-relations  of 
living  matter,  and  to  contribute  to  the  design  of  a  machine  in  which 
metabolism  takes  place.  The  proteinaceous  substances  may  in  plant 
protoplasm  form  a  widely  varying  proportion,  generally  very  low,  but 
sometimes  ranging  as  high  as  90  per  cent  of  the  entire  dry  weight  of 
the  protoplasm,  with  very  important  consequences  as  to  imbibition. 
The  enzymes  are  included  with  these  substances,  and  as  the  metab- 
olism, including  respiration  of  plants,  is  predominantly  a  complex  of 
transformations  in  carbohydrates,  the  possibilities  of  variation  in  this 
feature  are  very  great.  The  nature  of  the  pentose  derivatives  present 
in  the  protoplasm  may  also  be  a  feature  of  considerable  importance  in 
metabolism  and  water-relations,  as  suggested  by  the  differential  be- 
havior of  various  gums  and  mucilages  when  compared  with  that  of 
agar.  The.  colloidal  carbohydrates,  or  those  which  enter  into  the 
make-up  or  design  of  the  living  machine  and  the  proteinaceous  sub- 
stances, are  theoretically  mutually  nondiffusible,  so  that  the  gelation 
or  solidification  of  a  10  per  cent  solution  of  agar  and  gelatine  or  starch 
and  gelatine  according  to  Beijerinck1  would  result  in  a  mechanical 

1  Beijerinck,  M.  W.  Ueber  Emulsoidenbildung  wasseriger  Losungen  gewisser  gelatinierender 
Kolloide.  Zeitsch.  f.  Kolloid-Chem.,  7:16.  1910. 


Growth  and  Colloidal  Reactions.  3 

mixture  of  the  two  substances  in  which  the  two  would  exist  separately 
in  their  characteristic  emulsion.  The  ammo-acids,  on  the  other  hand, 
diffuse  readily  into  the  colloids,  and  these  may  be  visualized  as  being 
aggregated  with  the  carbohydrate  colloid,  in  both  phases,  and,  as  may 
be  seen  by  reference  to  Chapter  II,  they  set  up  water-relations  differ- 
ent in  some  features  from  those  determined  by  the  hydrogen  and 
hydroxyl  ions. 

The  place  of  the  lipins  in  the  hydration  mechanism  can  not  at  present 
be  made  the  subject  of  profitable  conjecture.  While  lecithin,  for 
example,  is  known  to  adsorb  water,  its  part  in  the  living  matter  of  the 
plant  must  be  all  but  negligible,  as  it  by  no  means  bulks  as  large  here 
as  in  the  animal. 

The  general  effect  of  the  salts  is  to  lessen  the  imbibition  capacity  of 
agar-protein  mixtures  when  in  simple  solutions  from  a  concentration 
of  2  N  to  0.00005  N.  Set  in  action  with  acids  or  in  antagonistic  rela- 
tions, other  effects  are  produced  in  sections  of  living  plants.  Although 
one  of  the  subjects  receiving  the  most  attention  in  colloidal  physics, 
and  although  extensive  experiments  with  tissues  and  cell-masses  of 
plants  and  animals  dealing  with  the  matter  have  been  made,  it  is  not 
yet  definitely  determined  whether  or  not  the  action  of  a  salt  upon  a 
biocolloid  may  be  expressed  by  the  algebraic  summation  of  its  acid  and 
base  as  originally  proposed  by  Pauli  for  gelatine,  or  whether  the  effect 
is  due  partly  to  other  factors. 

The  extent  to  which  protoplasm  may  be  made  up  of  dense  particles 
of  substances  of  a  single  category,  such  as  protein  or  globulin  granules, 
starch  grains,  or  of  minute  masses  of  more  highly  hydrated  strands  or 
globules  of  albumin  or  of  carbohydrates,  or  lipins,  or  of  combinations 
such  as  those  of  lipin  and  protein  in  the  mitochondria,  can  not  be 
visualized.  Neither  is  it  possible  to  say  whether  the  carbohydrate 
and  protein  molecules  are  equally  aggregated  in  both  the  continuous  or 
external  phase  and  the  discontinuous  or  internal  phase  of  the  gel,  or 
whether  one  predominates  in  each  phase  according  to  the  proportions 
in  which  they  are  combined.  In  any  case  phase  reversals  are  possible. 
The  imbibitional  reactions  of  the  living  matter  of  plants  are  seen,  how- 
ever, to  be  parallel  to  those  of  a  salted  carbohydrate-protein  gel  com- 
bined in  a  high  state  of  dispersion  and  questions  as  to  systemic  arrange- 
ment must  be  left  in  abeyance  for  the  present.  So  far  as  known,  it  is 
the  actual  composition  and  relative  proportions  of  the  substances  of 
the  main  organic  groups  and  the  amount,  the  stage,  and  sequence  of 
incorporation  of  the  infiltrated  salts  that  constitute  variables  of  the  first 
order  of  importance  in  the  determination  of  the  behavior  of  the  mass. 

The  chief  distinction  between  the  protoplasm  of  plants  and  that  of 
animals  may  be  taken  to  lie  within  the  play  of  these  major  features. 
Living  matter  in  animals  includes  lipins  and  consists  predominantly  of 
nitrogenous  material,  which  displays  maximum  hydration  capacity  in 


4  Hydration  and  Growth. 

a  hydrogen-ion  concentration  above  the  iso-electric  point,  or,  as  com- 
monly expressed,  when  in  a  state  of  acidosis,  in  consequence  of  which 
many  sweeping  premature  generalizations  have  been  made  as  to  the 
relations  of  electrolytes  to  protoplasm.  Plant  protoplasm,  in  so  far  as 
the  higher  forms  are  concerned,  is  poor  in  lipins,  is  usually  characterized 
by  a  major  proportion  of  carbohydrates,  although  in  such  simple  forms 
as  the  bacteria  the  protein  content  may  be  very  high.  The  water- 
relations  of  a  cell-mass  in  plants  will  naturally  be  determined  by  its 
protein-carbohydrate  ratio,  with  the  implied  corollary  that  a  varying 
hydration  capacity  is  displayed  which  may  reach  its  maximum  in  a 
condition  of  acidosis  in  forms  rich  in  nitrogen,  and  in  a  neutralized, 
relatively  salt-free  condition  in  those  in  which  the  proportion  of  col- 
loidal carbohydrate  is  relatively  great. 

Cytological  science  recognizes  that  homogeneous  states  of  the  col- 
loids do  not  prevail  throughout  the  cell  and  a  vast  literature  has  grown 
up  concerning  the  masses  of  unlike  composition,  structure,  and  form, 
some  of  morphological  value,  which  make  up  the  cell-body.  Attention 
has  naturally  been  concentrated  on  the  more  readily  visible,  durable, 
and  measurable  bodies,  some  of  which  are  indubitably  the  scene  of  per- 
formances of  the  first  rank,  and  form  the  chief  mechanism  in  genetics. 

It  is  not  to  be  forgotten,  however,  that  the  diverse  mixtures  of  gels 
and  sols  constituting  the  greater  part  of  the  protoplasmic  mass,  the 
structure  of  which  may  not  be  resolved  by  direct  microscopical  methods, 
is  the  ultimate  colloidal  machine  in  which  the  organs  of  the  cell  are 
built  up,  torn  down,  and  metamorphosed.  The  study  of  some  of  the 
solid  bodies  in  the  protoplasm  may  yield  the  same  comprehension  of 
the  play  of  chemical  energy  and  surface  tension  of  living  matter  as 
might  be  gained  of  the  cyclonic  forces  of  a  storm  by  a  measurement  and 
dissection  of  hailstones.  The  greatest  possibilities  in  cell  mechanics  are 
those  which  lie  in  the  changes  in  viscosity,  volume,  and  water-relations 
of  cell-organs  as  determined  by  the  composition  and  arrangement  of 
the  colloidal  emulsion  or  mesh  and  the  nature  of  the  metabolism  which 
goes  on  in  its  sols  and  gels. 

It  is  also  to  be  emphasized  that  it  is  not  only  untrue  but  unprofitable 
to  assume  that  the  living  matter  of  plants  and  animals  have  the  same 
general  chemical  properties.  The  difference  in  the  occurence  and  r61es 
of  lipin  in  the  two  groups  is  fundamental,  and,  in  addition  to  the  water- 
relations,  the  metabolisms  of  the  two  present  differences  not  attributable 
simply  to  the  carbohydrate-protein  ratio  in  their  composition.  Thus 
the  living  matter  of  plants  includes  within  its  metabolic  cycles  such 
features  as  the  synthesis  of  the  carbohydrates  and  of  the  amino-acids, 
the  last-named  capacity  being  exhibited  only  to  a  very  limited  extent 
by  animals,  which,  in  the  main,  are  characterized  by  a  mstabolism  of 
sugars  notably  different  from  that  of  the  plant. 


Growth  and  Colloidal  Reactions.  5 

Available  experiences  with  protoplasm  lead  to  the  conclusion  that 
it  may  be  considered  as  a  system  of  gels  and  sols  in  which  the  com- 
ponent material  is  found  in  different  conditions  with  respect  to  the  pro- 
portion of  water  combined  or  associated  with  it  (see  Chapter  II).  The 
more  fluid  parts  of  the  cell  owe  their  liquidity  to  the  fact  that  in  such 
material  water  containing  a  small  proportion  of  the  colloidal  material 
forms  a  medium  or  continuous  element  in  which  aggregations  of  mole- 
cules or  submicrons  combined  with  a  smaller  proportion  of  water  are 
dispersed  and  may  move  about  more  or  less  freely.  This  condition 
may  be  predicated  of  the  contents  of  the  vacuolar  cavities  and  of  the 
regions  in  the  cell  which  appear  clear  or  vacant  in  living  material  or  in 
cytological  preparations.  The  denser  parts  of  the  protoplast  would 
be  composed  of  a  much  larger  proportion  of  aggregated  matter  sepa- 
rated by  much  thinner  or  more  attenuated  layers  of  the  more  fluid  phase. 

Some  writers  assume  that  the  submicrons  of  colloidal  material  aggre- 
gate to  form  a  continuous  framework  or  structure  which  has  been 
likened  to  a  fine  sponge,  network  of  fibers,  or  honeycomb.  The  more 
liquid  colloid  fills  the  cavities  or  interstices  of  the  framework.  This 
condition  may  not  be  so  completely  the  reverse  of  the  preceding  as  to 
bring  the  more  fluid  colloid  into  complete  discontinuity.  It  may  be 
safely  assumed,  in  fact,  that  almost  any  mass  of  active  protoplasm  in- 
cludes both  conditions,  and  in  a  very  fluid  portion  of  the  living  matter 
small  fragments  of  gel  may  be  carried,  while  even  in  the  denser  newly 
separated  embryonic  cell-regions  minute  cavities  may  be  formed  by 
syneresis  in  which  the  colloid  is  in  its  extreme  disperse  condition.  Such 
syncretic  cavities  may  well  be  the  beginning  of  the  vacuoles,  in  contra- 
distinction to  the  view  which  assigns  a  definite  morphological  entity 
to  these  features. 

The  formation  of  these  syncretic  cavities,  the  size  of  the  molecular 
aggregates,  and  many  other  features  of  a  colloidal  mass  are  affected 
by  the  dilution  or  dispersion  of  the  original  material,  the  rate  of  dehy- 
dration and  gelation,  and  even  such  fundamental  characters  as  the 
relations  of  the  two  phases  may  be  affected  by  the  origin  and  rate  of 
deposition  of  the  material.  In  addition  to  these  very  fertile  sources 
of  variations  in  living  matter,  the  cell  at  most  times  carries  inclusions, 
such  as  starch  grains,  crystals,  and  protein  granules  which  are  com- 
paratively inert,  partly  by  reason  of  their  small  surfaces,  and  may  not 
exercise  much  influence  upon  the  surrounding  gel.  Oxidation,  proteo- 
lysis,  hydrolysis,  or  solution  of  these  bodies  may  set  free  or  split  the 
compounds  included,  and  these,  quickly  diffusing  through  the  colloidal 
mass,  may  play  an  important  part  in  the  morphological  crises  of  the  cell. 

Many  mistaken  attempts  have  been  made  to  compare  the  growth  of 
organisms  and  the  formation  of  crystals  directly,  and  to  establish  their 
identity  or  continuity.  The  results  of  such  efforts  serve  to  bemuse  the 
mystic,  to  divert  the  philosopher,  and  to  furnish  poetical  conceptions 


6  Hydration  and  Growth. 

to  writers  who  view  matter  and  all  material  conceptions  from  a  remote 
distance.  Colloids,  with  their  electrical  charge,  absorptive  and  adsorp- 
tive  properties,  and  molecular  arrangement,  display  a  series  of  char- 
acters fundamental  to  organic  growth,  of  which  swelling  as  a  result  of 
hydration  is  one  of  the  most  noticeable,  which  do  not  extend  to  crystals. 

Furthermore,  the  colloidal  systems  which  are  exemplified  in  living 
or  organic  material  are  rarely  at  rest  in  the  sense  of  which  this  may  be 
said  of  crystals.  It  is  true,  of  course,  that  substances  or  formations 
may  occur  in  nature  or  in  the  laboratory  which  are  made  up  of  both 
crystalline  and  colloidal  material,  and  it  is  also  true  that  some  com- 
pounds may  pass  from  one  condition  to  the  other,  but  the  action  by 
which  a  crystal  is  formed  is  not  one  coincident  with  colloidal  reactions, 
nor  does  the  perfect  crystal  behave  like  a  mature  cell,  organ,  or  organ- 
ism. In  fact,  the  more  perfect  a  crystalline  structure  may  be,  the 
farther  does  it  depart  from  the  state  in  which  it  might  display  activities 
or  enlargements  similar  to  those  of  growth  of  living  matter. 

The  essential  feature  of  an  idealized  growth  is  the  accretion  or  addi- 
tion of  water  and  material  to  the  mass  of  colloid  constituting  the  cell. 
The  actual  mechanism  of  incorporation  is  not  easily  delineated.  If 
protoplasm  consisted  of  a  system  of  colloidal  structures  such  as  those 
of  the  pentosans  and  the  proteins  interwoven  but  not  diffusing  into 
each  other,  the  more  solid  material  which  lowers  the  surface  tension  to 
the  greatest  extent,  having  the  least  attraction  for  water-molecules, 
would  tend  to  usurp  the  position  of  the  surface  layer.  Furthermore 
the  solid  phase,  whether  it  be  in  the  form  of  globules  or  in  the  continu- 
ous element,  would  tend  to  increase  and  crowd  together  with  a  lessen- 
ing of  the  more  liquid  phase.  This  would  imply  that  when  gelatine  in 
small  proportion  is  mixed  with  agar  or  starch  in  the  larger  proportion 
that  the  carbohydrate  would  form  the  colloidal  framework  or  mesh  as 
well  as  the  external  layer  of  the  mass.1 

The  separate  colloidal  masses  where  they  do  exist  have,  of  course 
definite  boundary  layers,  as  are  formed  wherever  two  colloidal  phases 
meet.  Protoplasm  may  not  be  regarded,  however,  as  altogether  a 
mechanical  admixture  of  minute  strands  of  material  of  different  com- 
position. Much  of  it,  including  the  more  fluid  portions,  must  consist 
of  molecules  of  carbohydrates,  proteins,  salts,  and  even  lipins  aggre- 
gated to  form  submicrons  in  the  disperse  phase  or  in  the  denser,  more 
solid  fibers,  mesh,  or  honeycomb  of  the  structure.  The  external  layer 
formed  might  well  be  in  a  sense  a  mosaic,  but  it  is  to  be  noted  that  no 
actual  proof  of  such  a  condition  is  at  hand.  Both  absorption  or  imbibi- 
tion and  osmosis,  including  differentiated  diffusions,  would  be  affected 
by  the  composition  and  relations  of  the  two  phases  of  the  colloids  in  this 
outer  layer,  and  it  seems  highly  probable  that  an  adequate  interpre- 

1  Free,  E.  E.  A  colloidal  hypothesis  of  protoplasmic  permeability.  The  Plant  World,  21 : 
141.  1918. 


Growth  and  Colloidal  Reactions.  7 

tation  of  permeability  will  be  obtained  by  a  study  of  these  features. 
Meanwhile  no  general  agreement  as  to  the  nature  of  the  "membrane" 
or  its  action  is  to  be  expected  until  many  widely  current  assumptions 
are  discarded.  The  external  layer  of  a  protoplasmic  unit  is  in  every 
case  a  product  of  the  surface  energy  of  the  mass  or  systems  of  living 
material  internal  to  it  and  of  the  medium,  and  has  no  other  permanent 
or  morphological  value.  Its  constitution  must  necessarily  vary  widely, 
as  does  that  of  the  living  protoplasm.1 

This  aspect  of  the  external  layer  is  one  which  finds  recognition  among 
writers  on  biophysics  in  various  ways.  Mathews  assumes  conditions 
in  the  protoplasm  which  are  not  valid  in  plants  when  he  says : 

"Thus  it  is  suggested  that  in  the  surface  of  contact  of  protoplasm  with 
water,  lipin  substances  will  accumulate  and  thus  make  a  kind  of  intermediate 
layer  of  a  lower  surface  tension  and  of  a  fatty  nature.  But,  inasmuch  as  the 
whole  substratum  of  the  cell  is  of  a  fatty  or  lipin  nature,  it  is  difficult  to  see 
how  the  surface  tension  of  the  junction  of  fat  and  water  could  be  changed  by 
the  passage  of  more  lipin  into  the  film;  and,  as  a  matter  of  fact,  there  is  no 
good  evidence  that  there  is  such  a  layer  about  the  protoplasm."1 

McClendon  recognizes  a  wider  range  of  facts,3  as  follows : 

"The  composition  of  the  plasma  membrane  remains  a  mystery.  It  seems 
logical  to  assume  that  its  building  stones  are  selected  from  the  chief  constitu- 
ents of  cells,  proteins,  fats,  lecithin,  cholesterin,  and  carbohydrates.  It  is  a 
very  unstable  structure,  as  will  be  shown  later." 

An  admirable  presentation  of  the  matter  is  to  be  credited  to  D'Arcy 
W.  Thompson,  the  keynote  of  which  lies  in  the  sentences:4 

"The  adsorbed  material  may  range  from  the  almost  unrecognizable  pellicle 
of  a  blood-corpuscle  to  the  distinctly  differentiated  'ectosarc'  of  a  protozoan, 
and  again  to  the  development  of  a  fully  formed  cell-wall,  as  in  the  cellulose 
partitions  of  a  vegetable  tissue.  In  such  cases,  the  dissolved  and  adsorbable 
material  has  not  only  the  property  of  lowering  the  surface  tension,  and  hence 
of  itself  accumulating  at  the  surface,  but  also  has  the  property  of  increasing 
the  viscosity  and  mechanical  rigidity  of  the  material  in  which  it  is  dissolved 
or  suspended,  and  so  of  constituting  a  visible  and  tangible  'membrane'." 

In  addition  to  the  external  layer  of  the  highly  hydrated  protoplasm, 
this  living  material  is  usually  separated  from  the  surrounding  medium 
by  walls  or  coats  of  specialized  character,  variously  formed,  and  which 
may  be  composed  in  part  or  altogether  of  material  originating  outside 
of  the  masses  which  they  inclose,  which  may  modify  the  diffusion  of 
liquids  into  the  colloidal  mass  in  a  very  important  manner. 

If  the  swelling  is  one  of  simple  hydration,  the  entrance  of  additional 
water  would  result  solely  in  an  increased  dispersion  of  both  phases  of 

1  See  Stiles  and  Jorgensen,  Quantitative  measurement  of  permeability.  Bot.  Gazette,  65 : 526.  1918. 

2  Mathews,  A.  P.     Physiol.  Chem.,  2d  ed.,  p.  211.     1916. 

3  McClendon,  J.  F.    The  physical  chemistry  of  vital  phenomena,  p.  95.    1917.    Princeton  Univ. 

Press. 

4  Thompson,  D'Arcy  W.     Growth  and  form,  pp.  281  and  282.     1917.    Cambridge. 


8  Hydration  and  Growth. 

the  colloid.  If  dissolved  salts  are  carried  by  the  water,  these  substances 
might  unite  chemically  with  the  material  in  the  molecular  aggregates 
in  both  the  more  liquid  and  the  more  solid  phases  of  the  colloid  and 
cause  changes  in  the  water-relations  of  the  mass,  or  the  dissolved  sub- 
stances entering  with  the  water  might  form  adsorption  compounds  by 
the  uniting  in  indefinite  proportions  with  the  colloidal  material  in 
which  the  water-relations  might  be  changed  in  another  way.  Such 
changes  would,  of  course,  be  followed  by  variations  in  volume.  It  is 
to  be  added  that  water  itself  may  enter  into  both  relations  with  the 
colloidal  material  and  that  the  initial  swelling  of  a  dried  colloid  prob- 
ably includes  such  a  chemical  combination  of  water  with  its  molecular 
aggregates.  The  action  of  salts  or  of  acids  brought  into  the  mass  with 
water  may  be  such  as  to  carry  the  dispersion  or  solvation  to  the  stage 
in  which  the  mass  assumes  a  liquid  condition.1 

In  that  type  of  growth  in  which  carbohydrates  or  proteins  are  carried 
into  the  mass  by  water,  it  may  be  seen  that  the  accumulation  of  the 
additional  material  in  the  more  liquid  phase  would  by  the  action  of  the 
forces  of  surface  tension  result  in  the  aggregation  of  new  masses  of 
material.  Such  formation  of  additional  elastic  gel  structure  might 
occur  theoretically  throughout  the  entire  mass  of  the  cell,  but  in  actual- 
ity would  be  modified  and  controlled  at  every  point  by  the  factors 
which  affect  hydration.  Aggregation  of  material  in  syncretic  cavities 
may  be  taken  to  present  possibilities  of  the  formation  of  specialized 
protoplasmic  masses  or  cell-organs  or  of  secretions.  Writers  with  a 
keen  historical  sense  may  be  disposed  to  see  in  the  conceptions  outlined 
above  a  modernized  statement  of  the  micellar  hypothesis  of  growth  of 
Naegeli,  but  no  great  interest  may  be  attributed  to  any  such  forced 
parallelism. 

The  measurements  described  afford  a  reliable  basis  for  the  conclusion 
that  the  extent  and  character  of  the  swelling  of  gels  compounded  of 
carbohydrates  and  proteins  or  protein  derivatives  depends  in  great 
degree  upon  the  proportions  of  the  main  constituents,  not  only  with 
respect  to  pure  water,  but  in  solutions  of  salts  and  electrolytes  in 
general.  The  general  effect  of  a  salt  on  hydration  depends  upon  its 
concentration  and  whether  it  is  already  present  in  the  colloid  in  chem- 
ical union  or  in  adsorption  with  the  colloidal  material,  or  whether  it 
enters  with  the  solution  or  water  of  hydration ;  also  upon  the  character 
of  the  salts  adsorbed.  The  extension  of  the  observations  upon  which 
these  conclusions  rest  to  living  cell-masses  and  to  desiccated  and  dead 
material  from  plants  demonstrates  that  colloids  may  be  compounded 
which  may  simulate  with  fair  parallelism  cell-colloids  with  varying 
carbohydrate-protein  ratio,  salt-content,  and  acidity.  In  no  feature 
is  this  more  striking  than  in  the  temperature  relations.  The  rate  and 
amount  of  swelling  of  plants,  and  of  colloidal  mixtures  which  simulate 

1Ostwald  and  Fischer.     Theoretical  and  applied  colloid  chemistry,  p.  101.     1917. 


Growth  and  Colloidal  Reactions.  9 

them,  in  water  or  in  some  solutions  may  increase  from  temperatures 
near  the  freezing-point  to  39°  to  46°  C.  and  then  fall  off  above  this 
region,  or  in  acid  solutions  of  a  concentration  normal  to  the  plant  both 
biocolloids  and  sections  of  living  and  dried  plants  may  show  decreased 
hydration  as  the  temperature  rises  above  17°  or  18°  C.1 

Nowhere  is  metabolism  more  active  than  in  the  embryonic  growing 
cell.  The  dissociations|which  are  usually  included  in  the  conception 
of  respiration  may  be  taken  to  concern  molecules  of  material  already 
present  in  the  more  liquid  phase  of  the  colloid  or  newly  introduced. 
The  splitting  of  the  sugars  results  in  the  formation  of  acids  as  one  stage 
of  the  process,  and  if  the  succeeding  stages  are  impeded  such  material 
accumulates,  acidosis  results,  with  new  temperature  relations  which 
may  affect  imbibition  and  the  enlargement  constituting  growth  in  a 
profound  manner.  Other  actions  will  depend  upon  the  composition 
of  the  cell  with  respect  to  its  carbohydrate  and  proteinaceous  constitu- 
ents. If  it  is  high  in  albuminous  material,  its  capacity  for  absorbing 
and  swelling  may  be  greatly  increased,  while  on  the  other  hand  this 
effect  will  be  greatly  lessened  if  a  large  proportion  of  pentosans  are 
present,  especially  in  the  presence  of  salts.  No  further  recapitulation 
of  detail  is  necessary  to  emphasize  the  fact  that  the  products  of  the 
reactions  within  the  cell  may  be  responsible  for  many  of  its  most  marked 
changes  in  behavior.  Such  changes  do  not  in  any  manner  give  support 
or  approval  to  vitalistic  theories  as  to  the  constitution  or  activities  of 
living  matter. 

The  other  soluble  carbohydrates,  including  the  hexoses — sucrose, 
dextrose — do  not  occur  in  the  cell  in  such  concentration  as  to  affect  the 
enlargement  of  the  protoplasmic  mass  directly,  but  in  the  vacuoles  they 
may  exert  an  osmotic  effect  additive  to  that  of  the  amino-acids  which 
may  accumulate  in  these  cavities.  It  is  to  the  osmotic  activity  of 
these  substances  in  the  vacuoles  that  turgidity  is  due,  and  a  by  no 
means  unimportant  part  in  the  maintenance  of  the  rigidity  of  organs 
and  other  features  is  to  be  ascribed  to  these  turgor  stresses  and  ten- 
sions. That  osmotic  pressure  may  also  play  an  important  part  in  the 
enlargement  of  the  plant  cell  may  well  be  concluded  from  the  fact  that 
in  the  stage  following  the  initial  swelling  of  the  embryonic  cell  a  large 
share  of  the  increase  in  volume  is  due  to  the  increase  of  the  vacuoles. 
The  inadequacy  of  osmotic  phenomena  and  of  the  conception  of  the 
semipermeable  membrane  to  provide  a  mechanism  for  the  trans- 
location  of  complex  material  from  cell  to  cell,  and  the  incorporation  of 
new  material  in  a  growing  mass  has  long  been  recognized.  It  would  be 
a  mistake  to  conclude  that  the  vacuole  is  simply  a  sac  charged  with 
electrolytes,  as  these  cavities  invariably  hold  proteins  and  carbohy- 
drates in  a  colloidal  condition  in  which  the  degree  of  dispersion  may 

1  MacDougal,  D.  T.    The  relation  of  growth  and  swelling  of  plants  and  of  biocolloids  to  temper- 
ature.    Proc.  Soc.  for  Exper.  Biol.  and  Med.,  15,  No.  3,  p.  48.     1917. 


10  Hydration  and  Growth. 

vary  widely,  but  still  absorb  water.  A  correct  delineation  of  the  man- 
ner in  which  osmosis  and  imbibition  interlock  in  growth  is  one  of  the 
tasks  demanding  the  immediate  attention  of  the  physiologist. 

It  has  been  assumed  in  the  present  work  that  when  the  chief  con- 
stituents of  protoplasm  are  brought  together  in  a  colloidal  condition, 
approximating  that  of  living  matter,  the  behavior  of  this  material  would 
furnish  data  fundamental  to  the  physics  of  growth.  The  justification 
for  this  assumption  is  to  be  found  in  the  following  pages,  in  which  are 
described  the  reactions  of  biocolloids,  of  dead  sections,  and  of  organs 
of  living  plants  in  hydration  and  growth  as  affected  by  solutions,  cul- 
ture media,  the  products  of  metabolism,  and  environmental  agencies, 
especially  temperature. 


II.    FUNDAMENTAL  FEATURES  OF  PHYTOCOLLOIDS. 


It  became  evident  in  the  earlier  stages  of  the  studies  described  in  the 
present  work  that  the  swelling  of  gelatine  does  not  afford  a  parallelism 
to  the  action  of  vegetative  cell-masses  of  the  higher  plants,  and  that 
only  in  certain  reproductive  elements  or  in  some  of  the  lower  forms  is 
the  proportion  of  nitrogenous  material  sufficiently  great  to  give  reac- 
tions similar  to  those  of  gelatine.  Experimental  demonstrations  of  the 
general  character  of  the  cell  colloids  was  first  made  with  sections  of 
joints  of  Opuntia.  The  results  of  an  extended  series  of  analyses  of 
these  plants  made  by  Dr.  H.  A.  Spoehr  covering  all  of  the  seasonal 
changes  were  available,  and  from  his  results  it  can  be  seen  that  their 
general  composition  is  about  as  shown  in  table  I.1 

TABLE  1. 


Constituents. 

Young 
joints. 

Old 

joints. 

Water  

p.  ct. 
95 

p.  ct. 
75 

Crude  protein  

0  5 

1  0 

Carbohydrates  hydrolyzable  with 
1  per  cent  HC1  

1.0 

10.0 

Cellulose  

1.0 

3.0 

Crude  fat  

0.25 

0.5 

Ash  

1.0 

3.5 

The  hydration  of  an  organ  or  cell-mass  with  a  composition  similar 
to  that  shown  in  table  1  would  of  course  be  determined  by  the  hydro- 
lyzable carbohydrates  and  proteins  and  affected  by  the  salts. 

The  first  experiments  were  directed  to  ascertaining  some  of  the 
reactions  of  the  carbohydrates  which  are  known  chiefly  or  entirely  in 
the  colloidal  form  and  which  might  be  a  constituent  of  the  plasmatic 
gels.  The  most  readily  available  representative  of  these  substances 
was  agar.  Strands  of  the  material  were  liquefied  in  water  at  60°  or 
70°  C.,  poured  into  shallow  molds  with  the  area  of  a  postal  card,  and 
allowed  to  desiccate  to  plates  from  which  pieces  3  by  5  mm.  were  cut. 
Swelling  was  measured  by  the  auxograph,  using  an  improved  form,  the 
essential  part  of  which  consists  of  a  compound  lever,  the  members  of 
which  are  pivoted  in  adjustable  bearings  in  a  rigid  brass  frame.2  The 
bearing  lever  has  a  forked  free  end  suitable  for  the  attachment  of  a 
counterpoise  and  of  a  vertical  swinging  arm  of  twisted  brass  wire. 
The  free  portion  of  this  vertical  lever  is  sheathed  with  a  section  of 

1  MacDougal  and  Spoehr.   Growth  and  imbibition.   Proc.  Amer.  Phil.   Soc.,  56  :  335.    1917. 
Philadelphia. 
*Ibid.,  327. 

11 


12  Hydration  and  Growth. 

glass  tubing  with  thin  walls,  drawn  to  a  point  and  sealed  hi  a  flame. 
The  pointed  glass  tip  fits  into  a  hole  in  the  center  of  a  thin  glass  plate 
resting  on  the  sections  to  be  swelled.  The  attachment  of  the  swinging 
lever  to  the  free  end  of  the  bearing  lever  may  be  adjusted  to  give  an 
amplification  of  the  swelling,  which  is  recorded  by  the  pen  tracing  an 
inked  line  on  a  sheet  of  paper  8  cm.  wide  ruled  to  millimeters.  The 
paper  is  coiled  inside  a  brass  cylinder  and  issues  through  a  slit  passing 
to  the  drum  of  a  clock  of  the  same  pattern  as  used  on  standard  thermo- 
graphs in  such  manner  as  to  present  a  uniform  plane  surface  to  the 
action  of  the  pen  in  all  parts  of  its  arc.  The  tautness  of  the  paper  may 
be  varied  by  altering  the  position  of  the  slit  in  the  brass  cylinder  and 
clocks  may  be  employed  which  give  the  paper  a  motion  of  28  cm.  in 
24  or  in  168  hours.  The  two  levers  are  connected  with  a  short  length 
of  jeweler's  chain  to  minimize  friction,  and  the  base  of  the  frame  carry- 
ing the  levers  is  seated  on  the  top  of  a  rack-and-pinion  column  with  a 
vertical  motion  of  12  cm.  and  is  capable  of  being  fastened  rigidly  at 
any  height  within  its  range  of  10  to  12  cm.  (fig.  1). 

The  dishes  finally  selected  for  containing  the  sections  to  be  immersed 
in  solutions  were  of  the  Stender  type,  5  cm.  in  diameter  and  24  mm. 
deep.  It  was  found  advisable  to  have  the  surface  of  the  bottdm  inside 
the  dish  ground  plane  in  order  to  avoid  slipping  and  movement  of  the 
swollen  sections  and  of  the  delicate  jellies  formed  by  the  biocolloids 
when  in  states  of  extreme  hydration.  For  similar  reasons  it  was  neces- 
sary to  place  the  entire  preparation  on  a  concrete  pier,  or  still  better 
upon  a  slab  of  marble,  granite,  or  concrete,  " floated"  in  a  large  box 
of  sandy  loam  which  had  a  direct  bearing  on  the  ground.  The  dishes 
containing  the  sections  were  seated  on  various  supports,  the  best  form 
being  that  of  an  iron  or  concrete  cylinder  about  12  cm.  in  height  and 
10  cm.  in  diameter.  Three  soft-metal  studs  were  let  into  the  basal 
end  of  this  cylinder  to  give  it  a  three-point  bearing  on  the  marble  slab. 
With  preparations  made  in  this  manner  and  with  the  counterpoise 
arranged  to  give  the  least  weight  upon  the  swelling  objects  compatible 
with  a  steady  following  movement  of  the  pen,  it  was  possible  to  obtain 
valuable  records  of  the  velocity  and  course  of  swelling  of  sections  of 
plants  and  of  various  colloidal  substances.  It  was  soon  learned  that 
more  reliable  results  might  be  obtained  with  thin  sections,  in  which 
the  coefficient  of  expansion  would  be  high  and  complete  hydration 
would  be  attained  quickly  and  with  less  dispersion  of  the  colloid  in 
the  liquid. 

No  records  of  temperature  in  the  earlier  tests  were  kept,  but  any  set 
of  swellings,  generally  of  three  or  four  different  solutions,  were  carried 
on  simultaneously  and  under  the  same  conditions.  It  is  to  be  under- 
stood, therefore,  that  in  tabulated  data,  as  table  2,  the  relative  swell- 
ing of  sections  of  one  sample  in  the  different  solutions  at  the  same  time 
only  may  be  compared.  The  experiments  had  not  been  carried  very 


Fundamental  Features  of  Phytocolloids.  13 

far  before  it  became  apparent  that  the  temperature  of  the  solution  in 
which  the  swellings  were  made  was  an  indispensable  feature  of  the 
control,  and  as  but  few  contemporary  workers  have  recorded  this  con- 
dition, it  is  not  possible  to  cite  their  results  with  profit.1  Some  inter- 
esting results  are  illustrated  by  the  figures  obtained  without  tempera- 
ture control,  which  derive  their  value  from  the  fact  that  all  of  those  in 


FIG.  1. — Auxograph  arranged  for  recording  changes  in  thickness  of  trio  of  sections  of  Opuntia  and 
of  biocolloids.  The  vertical  arm,  which  is  set  in  position  on  horizontal  arm  to  give  an  ampli- 
fication of  20,  rests  on  a  triangle  of  glass  laid  on  top  of  the  sections.  The  dish  containing 
the  sections  rests  on  an  iron  cylinder  to  secure  stability  and  a  weight  is  placed  on  the  T  base 
of  the  instrument.  The  record  sheet  is  ruled  to  millimeters  (not  shown)  with  heavier  hori- 
zontal lines  1  cm.  apart.  The  heavy  curved  lines  represent  hour  intervals.  The  space  is 
ruled  to  15-minute  intervals  (not  shown).  Height  of  clock  and  lever  supports  adjustable. 

1  MacDougal,  D.  T.     The  relation  of  growth  and  swelling  of  plants  and  biocolloids  to  tempera- 
ture.    Proc.  Soc.  Exper.  Biol.  and  Med.,  15  :  48.     1917. 


14 


Hydration  and  Growth. 


any  given  line  have  been  obtained  under  identical  conditions.  One  of 
these  comparative  series  made  in  1916  may  be  cited  as  an  example  of 
the  relative  behavior  of  agar  and  gelatine  to  water,  acids,  and  alkalies. 

TABLE  21. 


Swelling  of 
agar. 

Swelling  of 
gelatine. 

Sodium  hydroxid  (hundredth  molar)  .  .  . 
Hydrochloric  acid  (hundredth  molar)  .  .  . 
Water  

p.  ct. 
124 
113 
197 

p.  ct. 
250 
382 
83 

The  next  departure  in  the  experimentation  was  to  make  a  mixture 
of  these  two  substances  as  representing  the  carbohydrates  and  proteins 
of  the  plant,  and  this  was  done  in  a  series  of  plates  in  which  the  two 
elements  entered  in  proportions  from  1  or  2  to  9  parts  in  10.  The 
diverse  results  which  were  obtained  gave  ample  promise  of  affording 
many  useful  comparisons  with  the  action  of  plants. 

It  was,  of  course,  not  taken  for  granted  that  the  ammo-acids  used 
duplicate  those  which  are  found  in  the  plant  or  that  such  compounds 
afforded  all  of  the  more  important  factors  affecting  water-relations. 
The  next  step  in  the  making  of  a  colloidal  mixture  which  might  imitate 
the  action  of  the  plant  in  relation  to  water  was  to  use  various  albumin- 
ous compounds  to  furnish  the  nitrogenous  element  in  the  biocolloids. 

According  to  Beijerinck  and  others,  combination  of  agar  and  gelatine 
or  gelatine  and  starch  in  a  10  per  cent  solution  would  result  in  a  simple 
admixture  of  the  colloidal  masses  of  the  two  substances  in  the  form  of 
minute  masses  or  strands.2  Such  mixtures  would  be  more  intimate  and 
present  greater  surfaces  than  those  made  up  from  the  powdered  mate- 
rial brought  together  with  little  water  and  at  low  temperatures.  Pro- 
gressively finer  subdivision  of  the  materials  and  more  perfect  dispersion 
would  finally  reach  a  point  near  the  limits  of  gelation  where  the  sub- 
microns  of  agar  and  starch,  for  example,  might  come  together  in  the 
walls  or  fibers  and  in  the  more  liquid  part  of  the  two-phase  system  of 
colloids,  and  the  substances  in  the  parts  remaining  to  fill  cavities  or 
vacuoles  might  be  in  various  groupings,  according  to  one  view.  On 
the  other  hand,  very  weighty  theoretical  considerations  lead  to  the 
conclusion  that  the  relations  of  the  carbohydrate-protein  substances 
in  such  a  system  would  be  determined  quantitatively.  Thus  a  mixture 
of  8  or  9  parts  of  agar  and  1  or  2  parts  of  gelatine  or  albumin  at  a  high 
degree  of  dispersion  would  be  followed  by  a  gelation  in  which  the  pre- 
dominating substance,  agar,  would  form  the  external  or  continuous 

1  MacDougal,  D.  T.   Imbibitional  swelling  of  plants  and  colloidal  mixtures.  Science,  44  :  502. 
1916. 

2  Beijerinck,   M.   W.     Ueber  Emulsoidenbildung  bei  der  Vermischung  wasseriger  Losungen 
gewisser  gelatinierendern  Kolloide.     Zeitsch.  f.  Kolloid-Chem.,  7  :  16.  1910. 


Fundamental  Features  of  Phytocolloids.  15 

phase,  and  the  protein  the  internal  discontinuous  or  globular  phase. 
This  conception  is  a  very  attractive  one  because  of  the  possibilities 
implied.  Included  among  these  would  be  the  play  of  osmotic  forces 
in  the  inclosed  and  non-diffusible  gelatine,  which  might  be  a  contribu- 
tory factor  in  the  high  swelling  coefficients  displayed  by  biocolloids.1 

The  sections  and  plates  of  agar  and  proteins,  amino-acids,  etc.,  used 
in  the  accompanying  experiments  probably  included  the  materials  in 
many  possible  arrangements,  but  as  the  method  of  preparation  was 
uniform,  the  relative  value  of  the  results  obtained  from  them  remains 
unimpaired.  This  heterogeneity  is  a  direct  resultant  of  the  varying 
history  and  unequal  disposition  of  the  material  which  enters  into  the 
colloidal  mass,  and  would  find  direct  parallel  in  living  matter,  which  is 
practically  never  homogeneous  as  to  composition  or  uniform  as  to 
architecture  throughout  any  measurable  mass,  and  hence  its  morpho- 
logical units  are  not  isotropic  as  to  action  when  measured  with  com- 
mensurate accuracy. 

The  experimenter  dealing  with  the  hydration  of  these  elastic  gels 
does  not  proceed  far  before  he  becomes  aware  that  the  method  of  com- 
pounding, melting,  drying,  temperature,  and  other  features  of  the 
experience  of  the  biocolloids  influence  the  behavior  of  the  thin  plates 
which  may  be  made  from  them.  It  will  be  important,  therefore,  to 
describe  the  preparation  of  the  sections  which  were  used  in  these  and 
other  tests. 

The  agar  and  the  proteinaceous  material  should  all  be  from  one 
source  and  if  possible  from  a  single  lot  in  any  comprehensive  series  of 
tests  where  close  comparisons  are  desirable,  as  it  can  by  no  means 
be  assumed  the  composition  of  separate  lots  will  be  identical  as  to 
salt  or  nitrogen  content.  The  variations  in  gelatine  are  not  so  easily 
apprehended.  Both  agar  and  gelatine,  or  other  proteinaceous  com- 
pound used,  should  be  first  soaked  in  distilled  water  at  some  tempera- 
ture between  15°  and  20°  C.  for  a  period  of  a  half  hour.  The  agar  is 
now  heated  with  an  amount  of  distilled  water  over  a  water-bath  that 
will  bring  it  to  a  2.5  per  cent  solution  or  suspension.  The  suspension 
of  the  agar  may  be  accomplished  more  quickly  by  the  use  of  an  auto- 
clave. When  this  is  completed,  and  it  is  difficult  to  bring  the  last 
particles  into  liquid  form,  it  should  be  filtered  hot  into  a  flask  to  obtain 
a  clear  solution.  If  the  biocolloid  is  to  be  made  by  the  addition  of  an 
amino-acid,  any  one  of  these  substances  may  be  added  at  temperatures 
between  50°  and  80°  C.,  but  when  albumins  are  used  the  agar  solution 
must  be  cooled  in  a  warm  water-bath  until  it  comes  down  to  a  tempera- 
ture below  the  coagulation-point  of  the  latter  substances.  This  will 
be  found  somewhere  below  40°  C.,  and  the  protein  solution  should  be 
poured  in  with  sufficient  stirring  to  procure  good  admixture,  or  the 
same  end  reached  by  a  vigorous  shaking. 

1  See  discussion  in  Robertson,  T.  B.,  The  physical  chemistry  of  proteins,  pp.  294-350.    New 
York  and  London.     1918. 


16 


Hydration  and  Growth. 


The  most  useful  amount  of  material  for  making  dried  plates  of  a 
thickness  of  0.3  mm.  or  less  is  that  which  includes  10  grams  of  dry 
material  made  up  to  a  2  per  cent  liquid  or  solution.  This  amount  will 
form  two  plates  about  8  by  15  cm.  which  will  come  down  to  the 
desired  thickness  when  dried  at  15°  to  20°  C.  Two  methods  of  casting 
such  dried  plates  may  be  used,  according  to  the  composition  of  the 
colloid  that  is  being  manipulated.  Gelatine,  or  plant  mucilages,  or 
mixtures  in  which  these  substances  compose  half  or  more  of  the  whole, 
must  be  poured  directly  on  glass  plates  or  on  sheets  of  gold  or  plati- 
num foil. 

A  sheet  of  plate-glass  with  a  good  surface  is  set  in  a  level  position 
after  the  surface  has  been  well  cleaned  and  polished.  A  cell  about 
8  mm.  in  depth  and  10  by  15  cm.  is  now  made  on  it  by  glass  strips  fitted 
together  so  closely  than  when  the  warm  material  is  poured  onto  the 
glass  it  will  not  leak  out  at  joints  or  corners.  After  the  mixture  has 
cooled  sufficiently  for  the  gel  to  set,  the  plate  is  placed  in  the  desiccator 
and  drying  should  be  carried  on  at  such  rate  that  no  further  loss  of 
weight  occurs  after  about  40  hours.  The  dried  plate  is  now  worked 
free  from  the  glass  at  one  margin  by  an  instrument  with  a  chisel  edge 
and  then  stripped  free,  after  which  it  should  be  placed  in  a  closed  glass 
dish  to  keep  it  free  from  dust  and  undue  desiccation,  which  would 
produce  buckling  or  warping. 

FIG.  2. 

Drying  frame.  A  sheet 
of  wire  gauze  of  1  mm. 
mesh  stretched  on  a 
heavy  wooden  frame, 
being  fastened  secure- 
ly in  place  by  a  tightly 
fitting  strip  of  wood 
which  carries  the  mar- 
gin of  the  netting  down 
into  a  groove  in  the 
frame,  as  shown  in  the 
smaller  detail  drawing. 
A,  molding  form  of 
brass  bars;  B,  margin 
of  wire  netting  and 
clip  fastening  it  in 
place;  C,  wire  netting 
as  it  clears  frame.  The 
wire  netting  and  brass 
bars  are  coated  with 
fine  shellac. 

Many  plates  are  ruined  in  the  method  described,  and  when  possible 
the  following  devices  will  be  found  useful:  A  sheet  of  rustless  wire 
screen  with  a  mesh  less  than  2  mm.  is  stretched  on  a  heavy  wooden 
frame  (see  fig.  2),  so  as  to  offer  a  good  plane  surface.  A  mold  of  four 
brass  bars  is  laid  on  the  surface  and  a  sheet  of  hard  filter-paper,  such 
as  Whatman  No.  40,  is  fitted  into  this  cell.  The  frame  is  placed  in  a 
level  position  and  the  mixture  poured  into  the  shallow  cell  to  a  depth 
of  about  8  mm.  (fig.  3) .  After  it  has  cooled  and  set,  the  brass  members 


Fundamental  Features  of  Phytocolloids. 


17 


of  the  shallow  cell  are  removed  and  the  filter-paper  is  peeled  from  the 
margins  and  the  free  flaps  are  fastened  by  paste  to  the  edges  and  sur- 
faces of  the  wooden  frame  so  as  to  be  perfectly  taut  and  so  firmly  that 
when  the  colloid  dries  it  may  not  shrink  in  width  or  length.  Addi- 
tional care  should  be  taken  to  see  that  the  material  does  not  tear  loose 
from  the  paper  at  this  time,  and  if  it  does  it  should  be  secured  by  using 
some  fresh  material  as  a  paste  to  fix  it  to  the  paper.  The  margin  dries 
most  rapidly,  and  if  securely  attached  to  the  paper  holds  the  plate  in 
place  throughout  (see  fig.  4). 


FIG.  3. 

Drying-frame  with  sheet 
of  hard  filter-paper 
fitted  in  place  ready  to 
receive  liquid  colloidal 
mixture  which  is  to  be 
poured  on  the'paperto 
ajjtdepth  of  about 
8  to  10  mm. 


The  drying-frame  is  now  placed  on  a  rack  in  a  desiccator  which 
consists  of  an  inclosed  chamber  in  which  is  placed  an  electrically  driven 
fan  and  a  large,  shallow  pan  of  water.  The  best  results  are  secured  by 
a  rate  of  drying  which  results  from  having  air  with  high  humidity 
driven  over  the  plates  constantly  without  dehydrating  the  surface 
layers  too  rapidly  (fig.  5).  Drying  will  occupy  about  40  hours,  during 
part  of  which  time  it  may  be  advisable  to  stop  the  fan.  As  soon  as 
the  sheet  appears  to  be  dried  to  a  flexible,  leathery  consistency  the 
paper  may  be  freed  from  the  frame  and  then  stripped  from  the  plate 
of  material,  which  should  remain  plane,  with  but  little  curling  or  buck- 
ling. The  rough  and  uneven  margin  should  be  cut  away  with  scis- 
sors, the  data  as  to  composition,  etc.,  written  on  one  end  with  common 
ink,  and  then  placed  in  a  closed  dish  for  preservation  until  used. 

The  precautions  described  are  necessary  in  any  effort  toward  accuracy 
in  the  measurement  of  the  swelling  of  a  colloid,  as  the  increase  will, 
among  other  factors  in  its  experience,  reflect  most  strongly  the  method 
in  which  it  was  laid  down,  deposited,  or  dehydrated.  A  dried  section 
of  a  colloid  of  the  kind  used  in  these  experiments  tends  to  return  to  the 
dimensions  which  it  had  when  the  gel  set  or  cooled.  Standardization 


18 


Hydration  and  Growth. 


of  material  for  measurement  of  swelling  under  the  influence  of  various 
reagents  or  agencies  would  therefore  require  that  the  shrinkage  of  the 
material  as  it  dries  should  be  controlled.  This  may  be  done  in  many 
cases  in  the  manner  described.  Thus  in  the  case  of  biocolloids  con- 


Fio.  4. — Drying-frame  with  plate  of  colloid,  ready  to  be  put  into  the  desiccator.  The  molding- 
bars  A  have  been  removed  and  the  free  portions  of  the  filter-paper  have  been  carried  down 
over  the  side  of  the  frame  and  fastened  securely  in  place  with  paste. 


a 


FIG.  5. — Sectional  views  of  desiccator  for  drying  plates  of  colloids,  a,  frame  with  drying-plate 
lying  on  a  sheet  of  stretched  filter-paper,  which  in  turn  is  placed  on  a  shelf  of  slats;  6,  metal 
pan  containing  water  to  maintain  relatively  high  humidity;  c,  electric  fan  arranged  to  keep 
current  of  air  moving  over  the  suiface  of  the  water,  the  plate  of  colloid,  and  in  circulation  in 
the  chamber;  d  and  e,  hinged  lids  which  may  be  raised  to  control  ventilation  and  the  rela- 
tive humidity  of  the  chamber.  A  wooden  chamber  about  a  meter  in  height,  over  a  meter  in 
length,  and  80  cm.  in  width. 

sisting  of  9  parts  agar  and  1  part  bean  albumen  a  plate  cast  in  this 
manner  came  down  so  perfectly  that  a  strip  30  mm.  in  length  placed  in 
distilled  water  swelled  over  2,000  per  cent  in  thickness,  but  did  not 
increase  any  measurable  fraction  of  a  millimeter  in  length.  A  strip 


Fundamental  Features  of  Phytocolloids. 


19 


cut  from  the  extreme  margin  of  the  plate  would  doubtless  have  shown 
some  elongation. 

The  marginal  strip  of  a  plate  consisting  of  6  parts  agar,  3  parts  gum 
arabic,  and  1  part  gelatine  which  had  a  length  of  15  cm.  increased  to 
15.5  cm.  in  45  hours.  To  be  compared  with  this  elongation  of  3  per 
cent  is  that  of  the  increase  of  a  pile  of  sections  2  cm.  in  height  cut  from 
the  same  plate,  which  rose  to  15  cm.  in  24  hours,  showing  an  increase 
of  750  per  cent.  The  proportion  would  doubtless  have  been  still 
greater  had  the  swelling  of  one  section  been  measured  alone,  as  trios 
of  sections  cut  from  the  middle  of  the  plate  showed  swellings  of  1,141 
per  cent  at  14°  to  17°  C. 


FIG.  6. — Demonstration  of  the  swelling  of  sections  of  plates  of  agar  4  parts,  opuntia  mucilage 
4  parts,  gelatin  1  part,  bean  protein  1  part,  in  thickness  with  but  little  increase  in  length,  due 
to  the  manner  in  which  the  moist  colloid  was  held  while  drying. 

Another  test  of  the  same  kind  is  illustrated  by  figure  6.  Sections  of 
a  plate  consisting  of  4  parts  agar,  4  parts  opuntia  mucilage,  1  part  gela- 
tine, and  1  part  bean  protein  were  placed  in  a  frame  of  glass  rods  and 
immersed  in  a  vessel  of  distilled  water,  swelling  from  a  total  thickness 
of  15  mm.  to  a  total  of  180  mm.,  while  a  strip  cut  from  this  plate  which 
had  an  initial  length  of  8  cm.  had  elongated  to  8.5  cm.  The  increase 
in  thickness  was  1,200  per  cent,  while  that  in  length  was  but  6  per  cent. 

Plates  of  gelatine  invariably  showed  a  greater  increase  in  areal 
dimensions  than  that  of  biocolloids  such  as  those  mentioned  above,  and 


20  Hydration  and  Growth. 

no  plate  was  made  in  which  this  was  entirely  eliminated.  Thus  a  plate 
which  would  yield  increases  in  thickness  of  6  to  800  per  cent  in  trios  of 
sections  in  distilled  water  reached  a  length  as  much  as  50  per  cent 
greater  than  the  original  under  the  same  conditions.1 

A  unilateral  action  such  as  that  described  is  one  which  appears  to 
rest  upon  the  supposed  honeycomb  structure  of  the  colloid.  Dehydra- 
tion would  lessen  the  volume  of  the  mass,  and  as  the  sheets  or  strands 
of  denser  material  are  held  in  a  plane  parallel  to  the  surface,  the  spaces 
containing  the  more  discontinuous,  more  liquid  element  would  be 
deformed  and  their  vertical  diameter  decreased.  Accession  of  liquid 
or  of  water  would  be  followed  by  the  partial  resumption  of  the  original 
form  and  dimensions.  Experience  in  dealing  with  a  large  number  of 
plates  leaves  the  impression  that  the  swelling  does  not  bring  the  sec- 
tions back  to  the  thickness  of  the  cooled  gel  as  it  was  originally  in  the 
mold. 

The  fact  that  colloids  such  as  those  present  in  living  matter  may 
retain  a  shearing  strain  was  recognized  by  Butschli  and  was  the  subject 
of  some  experimentation  by  Hardy2  in  a  study  of  coagulation  phe- 
nomena, who  concludes  that  "shearing  a  colloidal  mass,  fluid  or  solid, 
actually  does  produce  heterogeneity  or,  simply,  structure,  which  is 
fixed  by  the  process  of  coagulation."  Such  sheared  masses  of  colloid 
are  doubly  refractive.  Klocke  demonstrated  the  acquisition  of  such 
double  refraction  by  sheets  of  gelatine  which  were  dried  on  frames 
covered  with  tin-foil.  It  is  to  be  noted  that  the  plates  of  gelatine 
which  were  dried  without  superficial  shrinkage  in  my  own  experiments 
when  hydrated  showed  some  extension,  while  those  of  the  agar-protein 
mixture  did  not.  The  hydration  in  both  cases  presumably  removed 
the  strain  as  the  structure  produced  by  the  stress  disappeared.  Great 
cytological  interest  attaches  to  the  simple  experiment  by  Hardy,  in 
which  a  small  quantity  of  a  colloidal  solution  is  drawn  along  a  glass 
slide  by  the  point  of  a  needle,  after  which  it  is  "fixed"  by  the  methods 
of  the  cytologist,  with  the  result  that  the  mass  appears  to  consist  of  a 
number  of  fibrillaB  "  *  *  *  so  striking  that  they  look  as  if  one  might 
isolate  them  by  teasing." 

Much  interest  also  attaches  to  some  recent  work  of  Miss  C.  L.  Carey 
of  Barnard  College  upon  the  structure  of  agar  films.  2.5  per  cent 
gels  of  this  substance  were  prepared  by  a  method  similar  to  that 
described  on  page  16  for  preventing  superficial  shrinkage.  When  such 
plates  were  dried  at  45°  to  70°  C.  and  again  placed  in  water  the 
rehydrated  plates  yielded  drops  of  water  so  readily  that  an  examination 
of  thin  sections  under  the  microscope  was  made,  revealing  cavities 

'For  the  original  notice  of  increase  in  thickness  and  not  in  length,  see  MacDougal  and  Spoehr, 
Growth  and  Imbibition,  Proc.  Am.  Phil.  Soc.,  56  :  343,  344.  1917.  Philadelphia. 

1  Hardy,  W.  B.  On  the  structure  of  cell  protoplasm.  Journal  of  Physiol.,  24  :  158.  1899. 
See  especially  pp.  187-190.  London. 


Fundamental  Features  of  Phytocolloids. 


21 


with  their  long  axes  parallel  to  the  surface,  as  shown  in  figure  7,  which 
was  furnished  by  Miss  Carey  in  response  to  the  request  of  the  author. 

The  extraction  and  preparation  of  a  number  of  substances,  includ- 
ing protein  from  oats,  albumin  and  globulin  from  beans,  the  total  pro- 
tein of  beans,  asparagin,  aspartic  acid,  etc.,  was  undertaken  by  Dr. 


FIG.  7. — Longitudinal  section  of  an  agar  plate  dried  at  70°  C.  without  superficial  shrinkage,  with 
development  of  elongated  spaces  or  cavities  which  are  found  when  the  film  is  hydrated 
Drawn  with  camera  lucida  by  Miss  C.  L.  Carey.  X78.5  diam. 


FIG.  8. — Scale  designed  for  measuring  thickness  of  paper  and  suitable  for 
determiningjjthickness  of  sections  of  plates  of  biocolloids.  Sheets  of  a 
thickness  of  0.001  to  0.11  inch(=  2.8  mm.)  may  be  measured  (see fig.  23 
for  calipers  used  in  measuring  larger  objects). 

Isaac  Harris,  of|Squibb  &  Sons'  laboratories,  New  Brunswick,  New 
Jersey,  while  Mr.  E.  R.  Long  furnished  preparations  of  such  sub- 
stances as  zein,  which  made  it  possible  to  make  up  plates  of  biocolloids 
entirely  from  products  of  plants. 

The  swelling  of  mixtures  of  agar  and  of  the  protein  extract  of  bean 
in  plates  0.3  to  0.4  mm.  in  thickness  were  as  shown  in  table  3. 


22 


Hydration  and  Growth. 


The  greatest  capacities  for  hydration  encountered  were  those  in 
which  plant  proteins,  such  as  those  of  oats,  were  added  to  agar  in  the 
proportion  of  about  1  in  10,  a  ratio  which  finds  its  equivalent  in  the 
constitution  of  many  of  the  higher  plants.  The  maxima  exhibited  by 
such  mixtures  are  not  duplicated  by  the  plant,  in  which  the  presence  of 
salts  in  the  colloids  and  the  morphological  structure  operate  to  limit 
the  amplitude  of  the  swelling. 

TABLES. 


Dist.  water. 

Hydrochloric 
acid, 
0.01  M. 

Sodium  hydrox., 
0.01M. 

Gelatine  90,  protein  10  I 
(Phaseolus)  ] 

p.  ct. 
585 
486 

p.  ct. 
1,401 
1,200 

p.  ct. 
942 
704 

386 

800 

Averages  

485 

1,300 

817 

Gelatine  75,  protein  25  f 
(Phaseolus)        \ 

696 
500 

818 
1  ,  060 

621 

848 

Averages.  ....... 

598 

939 

734 

Agar   90,    protein    10  f 
(Phaseolus)  \ 

800 
800 

50 
75 

150 
150 

Averages  

800 

62 

150 

Agar  99,  protein  1  f 
(Phaseolus)  \ 

1,080 
800 

300 
360 

220 
240 

Averages  

940 

330 

230 

The  method  of  admixture  of  the  carbohydrate-protein-saline  con- 
stituents of  the  biocolloids  consisted  mainly  in  the  use  of  temperatures 
which  would  bring  all  of  the  substances  into  a  liquid  condition  in  which 
they  might  be  as  intimately  united  as  possible,  and  the  plates  formed 
appeared  translucent  but  uniform  throughout,  although  it  is  not  to  be 
assumed  that  the  components  in  this  case  or  in  any  preparation,  or  in 
protoplasm,  are  mutually  interdiffused.  It  seemed  desirable  to  attempt 
to  make  mixtures  in  which  the  carbohydrate-protein  elements  would 
be  less  intimately  united,  and  to  that  end  some  simple  experiments 
with  powdered  agar  and  powdered  gelatine  were  carried  out. 

Powdered  gelatine  and  agar  which  would  pass  the  millimeter  mesh 
of  a  screen  were  used  to  ascertain  what  degree  of  expansion  would  be 
registered  on  the  auxograph  when  these  were  simply  placed  in  a  layer 
in  the  dish  and  subjected  to  the  action  of  solutions.  Whatever  the 
arrangement  of  the  material  in  these  particles,  their  separate  action 
in  swelling  and  in  dispersion  or  solution  would  be  free  from  the  action 
resulting  from  structures  such  as  those  presented  by  plates  held  rigidly 


Fundamental  Features  of  Phytocolloids.  23 

while  being  dried.  When  a  layer  of  powdered  gelatine  was  placed  in 
the  bottom  of  a  Stender  dish  to  a  depth  of  1.5  mm.  and  covered  with  a 
perforated  glass  triangular  plate  which  would  go  into  the  dish  readily, 
a  swelling  of  270  per  cent  in  water  at  18°  C.  was  registered  by  the  auxo- 
graph.  Furthermore,  this  increase  was  not  the  rapid  swelling  of  a 
mass  with  subsequent  relaxation,  but  lasted  over  5  days;  the  greater 
part  of  the  swelling  occurred  during  the  first  half  hour,  then  continued 
at  a  decreasing  rate  for  the  period  mentioned. 

A  similar  experiment  was  made  with  powdered  agar,  the  particles  of 
which  were  probably  of  a  much  smaller  average  size.  The  swelling 
was  of  the  same  kind,  but  was  complete  in  4  days,  although  reaching 
the  higher  total  of  317  per  cent.  This  higher  hydration  capacity  is 
characteristic  of  agar  as  compared  with  gelatine. 

It  is  of  course  to  be  expected  that  when  two  colloidal  substances  in  a 
two-phase  system  are  combined  and  the  resulting  material  is  subjected 
to  agencies  that  will  coagulate  or  neutralize  one  of  them,  the  section 
would  then  show  the  relations  of  the  one  still  in  the  colloidal  state. 
This  was  demonstrated  with  some  completeness  by  a  mixture  of  agar 
and  milk  albumin. 

Preparations  in  which  1  part  albumin  from  milk  was  stirred  into 
9  parts  melted  agar  at  40°  C.  and  under,  thus  remaining  active  and 
suspended,  showed  swellings  in  the  form  of  dried  plates  0.1  to  0.15  mm. 
in  thickness  at  16°  C.,  as  shown  in  table  4. 

TABLE  4. 

p.  ct. 

Water 1,792 

Citric  acid,  0.01  N 333 

Sodium  hydroxid,  0 . 01  M 386 

The  high  swelling  in  water,  the  increased  imbibition  in  acid,  and  the 
equalization  of  the  acid  and  alkali  effects  are  characteristic  of  agar- 
protein  mixtures  and  are  in  contrast  with  the  reactions  of  the  following 
test,  in  which  the  albumin  was  coagulated.  As  a  result  it  no  longer 
intermeshed  with  the  agar  in  the  gel,  but  aggregated  as  small  particles, 
indifferent  to  the  presence  or  proportion  of  water.  A  mixture  of  agar 
95  parts  and  milk  albumin  5  parts  was  prepared,  in  which  the  last- 
named  substance  dissolved  in  water  was  added  to  the  melted  agar  at 
a  temperature  near  100°  C.,  at  which  coagulation  followed.  The  mix- 
ture, however,  when  poured  on  a  glass  plate,  dried  into  a  film  about 
0.13  mm.  in  thickness,  which  had  a  leathery  texture  and  was  trans- 
parent and  appeared  homogeneous.  Sections  swelled  under  the  auxo- 
graph  at  16°  C.  increased  261.5  per  cent  in  distilled  water,  346.2  per 
cent  in  hundredth-molar  sodium  hydroxid,  and  but  191.2  per  cent  in 
hundredth-normal  citric  acid.  The  proportions  are  in  general  accord 
with  those  obtained  by  swelling  of  agar  alone,  suggesting  that  the 
neutralized  or  coagulated  albumen  has  no  effect  on  the  imbibition 
capacity  of  agar,  in  which  it  may  be  incorporated. 


24  Hydration  and  Growth. 

Other  substances  were  next  tested  which  are  insoluble  in  water  and 
hence  might  not  be  expected  to  enter  into  the  two-phase  system  with 
agar.  Mr.  E.  R.  Long  prepared  some  zein,  a  protmaine  derived  from 
Zea  mais,  at  his  laboratory  at  Seattle,  Washington,  early  in  July  1917, 
and  this  was  made  up  with  1  part  of  zein  to  9  of  agar. 

The  fine,  iregular  particles,  after  being  wetted,  were  stirred  into  the 
melted  agar  and  the  mixture  was  poured  onto  a  glass  slab  and  dried 
down  to  a  thickness  of  about  0.3  mm.  The  plate  was  rough  to  the 
touch,  the  granular  particles  of  the  zein  being  distinctly  visible  as 
opaque  masses.  Sections  tested  under  the  auxograph  gave  the  meas- 
urements as  follows: 

TABLE  5. 

p.  ct. 

Water 1,033.3 

Citric  acid,  0.01  N 400 

Sodium  hydroxid,  0.01  M 350 

The  presence  of  the  zein  could  not  be  said  to  be  entirely  without 
effect,  as  these  measurements  show  some  departure  from  agar  in  the 
relatively  high  swelling  in  acid.  The  imbibition  in  the  hydroxid  solu- 
tion was  extremely  slow.  Saturation  was  reached  in  24  to  30  hours  in 
water  and  acid,  but  enlargement  continued  for  twice  this  period  in 
hydroxid.  All  liquids  were  renewed  at  36  hours.  An  acceleration  ensued 
in  hydroxid  and  the  swelling  was  still  in  progress  at  the  end  of  48  hours, 
at  which  time  the  measurements  were  as  above.  Some  of  the  equaliza- 
tion or  increase  in  the  swelling  of  the  biocolloid  in  acid  and  its  con- 
tinued swelling  in  hydroxid  is  probably  due  to  the  fact  that  zein  is 
slightly  soluble  in  both  acids  and  alkalies. 

The  addition  of  1  part  globulin  from  beans  to  9  parts  agar  resulted 
in  the  formation  of  dried  plates  much  like  those  of  agar-zein.  The 
globulin,  not  being  soluble  in  water,  was  incorporated  as  small  globular 
masses.  Swellings  of  sections  0.2  mm.  in  thickness  were  exhibited  as 
shown  in  table  6,  at  16°  C. 

TABLE  6. 

p.  ct. 

Distilled  water 875 

Potassium  nitrate,  0.01  M 575 

Potassium  nitrate,  citric  acid,  0. 01  N 550 

Citric  acid,  0. 01  N 525 

Potassium  nitrate,  potassium  -hydroxid,  0.01  M 450 

Potassium  hydroxid,  0.01  M 300 

The  proportionate  imbibition  in  water,  acid,  and  hydroxid  is  one 
characteristic  of  agar  with  a  small  proportion  of  protein.  The  solubility 
of  globulin  in  salt  solutions  would  lead  to  the  expectancy  that  its  presence 
would  result  in  a  modification  of  the  swelling  of  agar  in  saline  solutions.1 

The  " bean-protein"  which  has  been  used  so  extensively  in  these 
experiments  is,  as  noted  elsewhere,  an  extract  with  water  in  which  the 

1  See  Zsigmondy,  Behavior  of  globulins,  in  chemistry  of  colloids,  p.  222.     1917. 
Robertson,  T.  B.     The  physical  chemistry  of  proteins.     1918.     New  York. 


Fundamental  Features  of  Phytocolloids.  25 

albumin,  and  some  of  the  globulin,  is  dissolved  in  the  water,  which  also 
contains  the  salts  of  the  bean.  The  effects  of  albumin  were  tested  sepa- 
rately, following  the  measurement  of  globulin  effects,  and  the  swellings 
of  thin  plates  of  9  parts  agar  and  1  part  albumin  were  as  follows : 

TABLE  7. 

p.  ct. 

Distilled  water 1 , 158 

Potassium  nitrate,  0.01  M 947 

Potassium  nitrate,  citric  acid,  0 . 01  N 500 

Citric  acid,  0.01  N 421 

Potassium  nitrate,  potassium  hydroxid,  0.01  M 421 

Potassium  hydroxid,  0.01  M 316 

The  comparison  of  the  above  data  with  those  obtained  from  the  agar- 
globulin  reveals  the  fact  that  while  the  globulin  does  not  appear  to 
increase  the  imbibition  capacity  of  agar  very  much,  the  albumin  does 
exercise  such  positive  effect,  the  mixture  showing  a  capacity  three 
times  as  great  as  in  acid  or  alkali.  The  swelling  in  acids  is  slightly 
greater  than  in  alkalies,  in  accordance  with  ti  e  action  of  other  mixtures 
of  albumin.  Imbibition  by  the  globulin  mixture  in  potassium  nitrate  is 
relatively  high  compared  to  water-effects,  while  it  scarcely  rises  above 
that  in  acids.  The  swelling  of  the  agar-albumin  in  this  salt  is  more 
than  twice  that  of  acidified  and  alkaline  salts,  acids,  and  alkalies. 

The  presence  of  insoluble  inclusions  is  of  course  the  normal  and  usual 
condition  in  the  cells  of  plants  during  extended  periods,  and  it  was 
desirable  to  ascertain  whether  or  not  material  wholly  neutral  in  biocol- 
loidal  plates  would  affect  hydration.  The  first  trial  was  made  with  a 
cotton  lace  about  1  cm.  in  width  and  0.5  mm.  in  thickness.  This  was 
well  softened,  and  when  laid  in  the  mold  the  warm  colloidal  mass  was 
poured  over  it,  accomplishing  an  intimate  penetration  among  the 
smaller  fibers  of  the  threads. 

The  portion  of  a  plate  of  agar  and  oat  protein  free  from  the  webbing 
dried  down  to  a  thickness  of  0.18  mm.,  and  sections  of  this  swelled 
2,111  per  cent  in  distilled  water,  while  the  increase  in  swamp  water 
was  1,277  per  cent.  Sections  containing  webbing  swelled  1,583  per 
cent  in  bog  water  and  the  same  amount  in  distilled  water,  and  1,195 
per  cent  in  swamp  water,  calculated  on  the  basis  of  the  thickness  of 
biocolloid  noted  above.  It  is  by  no  means  certain,  however,  that  the 
colloid  does  dry  in  equal  mass  on  the  webbing.  (See  Chapter  VI  for 
discussion  of  bog  water.) 

Calculated  in  terms  of  actual  thickness,  the  swelling  of  the  webbed 
sections  was  491  per  cent  in  bog  water  and  in  distilled  water,  while  it 
was  but  371  per  cent  in  swamp  water.  The  presence  of  the  webbing 
appears  to  diminish  the  proportion  of  swelling  in  distilled  water  and 
bog  water,  but  not  that  in  swamp  water.  Swamp  water  (see  p.  68) 
contains  an  amount  of  calcium  salts  which  notably  affects  swelling  in 
clear  sections.  This  operated  to  mask  any  effect  due  to  the  presence 
of  the  cotton  fibers  in  colloidal  sections. 


26  Hydration  and  Growth. 

Still  another  type  of  inclusion  was  tested  by  the  incorporation  of 
spores  of  Lycopodium  in  liquefied  agar.  These  spores  are  not  readily 
wetted,  and  hence  they  could  be  worked  into  a  colloidal  mixture  only 
at  low  temperatures,  when  it  was  in  a  stage  nearing  gelation. 

The  first  attempt  was  one  in  which  2.5  parts  by  weight  of  spores  were 
mixed  with  40  parts  by  dry  weight  of  agar.  The  agar  was  liquefied  in 
the  usual  manner,  and  when  it  had  come  down  to  a  temperature  of 
about  40  was  strained  through  cheese  cloth  into  a  beaker.  The  quan- 
tity of  spores  given  above  was  now  placed  on  the  surface  and  the  whole 
was  vigorously  stirred  for  several  minutes  with  an  ordinary  revolving 
egg-beater.  The  agitation  was  continued  until  the  temperature  fell 
to  35°  C.,  when  the  whole  was  cast  as  a  plate  in  the  usual  manner.  The 
dried  plate  was  0.3  mm.  in  thickness  and  showed  the  spores  and 
numerous  clumps  of  spores  embedded  in  it,  with  very  few  really  coming 
to  the  surface.  When  sections  of  the  ordinary  size  were  cut  and  swelled 
at  18°  C.  they  showed  some  buckling.  The  swellings  hi  distilled  water 
and  in  asparagin  0.05  M  were  equivalent,  being  850  per  cent  in 24  hours, 
with  some  increase  still  in  progress,  the  rate  being  greater  in  distilled 
water. 

The  second  test  was  arranged  with  sections  0.27  and  0.28  mm.  in 
thickness,  which  swelled  1,125  per  cent  in  0.01  M  asparagin  and  667 
per  cent  in  acetic  acid  0.01  N,  both  pairs  of  tests  showing  an  expansion 
far  less  than  might  be  expected  from  the  agar  alone.  It  seems  quite 
safe  to  conclude  that  inclusions  such  as  bodies  of  zein,  globulin,  coagu- 
lated albumin,  fine  threads  of  glass,  cotton  fibers,  and  spores  lessen  the 
hydration  capacity  of  the  gel  in  which  they  may  be  embedded.  As 
their  effects  are  due  directly  to  the  area  of  surface  and  radius  of  curva- 
ture, the  action  of  a  comparatively  small  amount  of  finely  divided 
material  would  be  very  much  greater  in  the  cell.  The  foregoing  results 
are  in  accordance  with  -those  of  Hardy,  who  found  that  solids  included 
in  a  colloid  before  fixation  may  influence  the  structure  of  films  in  a 
material  manner.  Grains  of  carmine  incorporated  in  liquid  colloids 
modified  the  mesh  and  the  thickness  of  the  plates  or  bars  or  more  solid 
material,  and  the  prevalence  of  insoluble  particles  in  plant  cells  renders 
such  observations  of  great  interest.1  * 

1  Hardy,  W.  B.    On  the  structure  of  cell  protoplasm.     Journal  of  Physiology,  24:  158.  1899.   See 
p.  186. 


III.    THE  CONSTITUENTS  OF  BIOCOLLOIDS  WHICH  AFFECT 
HYDRATION  AND  GROWTH. 

Some  organs  and  cell-masses  of  plants  as  well  as  of  animals  display 
swelling  reactions  similar  to  those  of  gelatine  with  respect  to  acids  and 
salts,  and  a  great  deal  of  discussion  of  the  colloidal  action  of  proto- 
plasm has  been  based  on  the  assumption  that  this  parallelism  runs 
throughout.  That  investigation  should  have  taken  this  course  is 
natural  when  it  is  recalled  that  gelatine,  in  common  with  proteins  and 
many  protein  derivatives,  is  amphoteric,  and  may  dissociate  either  as 
an  acid  or  as  a  base,  being  stronger  as  an  acid  than  as  a  base.  In  a 
condition  of  neutrality  or  at  its  iso-electric  point  its  hydrogen-ion  con- 
centration is  represented  by  the  symbol  pH  =  4.7.  It  is  to  be  seen 
that  the  diversity  of  hydration  reactions  which  such  substances  may 
display  might  well  give  rise  to  the  assumption  in  question.  Further- 
more, it  is  to  be  granted  that  some  organs  and  cell-masses  of  animals  as 
well  as  of  plants  are  so  high  in  nitrogenous  compounds  as  to  be  charac- 
terized by  the  reactions  of  amphoteric  colloids.  Thus,  for  example,  bac- 
teria may  contain  so  much  nitrogenous  material  that  the  dried  remainder 
obtained  from  them  may  appear  to  consist  principally  of  albumin.1 

It  would  be  a  mistake,  however,  to  assume  a  close  identity  of  the 
protoplasmic  machine  as  to  its  colloidal  components.  The  results 
described  in  the  present  work  make  it  evident  that  it  is  a  heterogeneous 
gel,  which,  in  plants  at  least,  is  largely  composed  of  inert  or  neutral 
carbohydrates  of  the  pentosan  group,  of  which  agar,  gum  arabic,  and 
mucilages  are  examples.  The  swelling  or  hydration  of  such  gels  is 
modified  by  the  action  of  the  proteins,  amino-acids,  and  other  nitro- 
genous substances  which  may  be  incorporated  with  them,  but  through- 
out all  of  my  experimentation  it  was  evident  that  the  water-relations 
of  growing  plants  were  more  of  the  character  of  pentosans  than  of 
gelatines. 

Reproductive  cells  and  elements  of  all  kinds  may  be  expected  to 
prove  high  in  nitrogen,  and  hence  would  show  swelling  reactions  of  the 
general  nature  of  gelatine.2  So  well  is  this  established  that  is  is  pos- 
sible to  predict  the  main  facts  as  to  nitrogen-content  upon  the  basis  of 
a  series  of  swelling  tests  with  distilled  water,  acids,  and  alkalies. 

The  relation  of  the  nitrogen-content  to  swelling  is  well  illustrated  by 
some  reactions  of  red  algae  of  the  Pacific  Coast  which  were  studied  at 
the  Coastal  Laboratory,  Carmel,  California,  in  July  and  August  1917, 
by  Dr.  J.  M.  McGee.3 

1  Thompson,  D.  A.  W.     Growth  and  form,  pp.  40  and  41.     1917.     Cambridge  Univ.  Press. 

1  Lloyd,  F.  E.    Colloidal  phenomena  in  the  protoplasm  of  pollen  tubes.     Report  Dept.  Bot. 
Research,  Carnegie  Inst.  Wash,  for  1917  (Year  book  No.  16).     1918. 

3  McGee,  J.  M.  The  imbibitional  swelling  of  marine  algse.  Plant  World,  21  :  13.  1918.  Balti- 
more. 

27 


28  Hydration  and  Growth. 

Trios  of  sections  of  the  laminae  were  swelled  in  various  solutions  and 
their  increase  registered  by  the  auxograph.  These  marine  algae  have  a 
normal  balance  enabling  them  to  exist  in  sea-water  which  contains 
about  3.50  per  cent  total  salts.  The  effect  of  the  various  substances 
on  imbibition  in  these  plants  was  therefore  obtained  by  adding  them  to 
sea-water  in  such  quantities  that  they  formed  hundredth-molar  solu- 
tions. The  results  with  Iridcea  laminarioides  were  as  follows  at  16°  C. : 

TABLE  8. 

Thickness,  0.4  mm.  p.  ct. 

Sear-water  +  NaOH,  0.01  M 25 

Sea-water  +  HC1,  0.01  M 31 

KNO3   +  citric  acid,  0.01  N 175 

Young  fronds  of  Gigartina  exasperata  gave  average  swellings  as 
below  at  16°  C.: 

TABLE  9. 

p.  ct. 

Sea-water,  sodium  hydroxid,  0 . 01  M 28 

Sea-water,  hydrochloric  acid,  0.01  M 38 

Potassium  nitrate,  citric  acid,  0 . 01  N 142 

Such  reactions  are  indicative  of  a  high  proportion  of  amino-acids, 
which  probably  fell  off  toward  maturity,  and  which  may  have  been 
extracted  by  washing  as  sections  which  had  been  treated  with  distilled 
water,  a  treatment  which  would  result  in  the  extraction  of  some  of  the 
salts  and  the  amino-acids.  Such  sections  when  dried  to  a  thickness 
of  0.5  mm.,  gave  swellings  at  16°  C.  as  follows: 

TABLE  10. 

p.  ct. 

Distilled  water 4,331 

Hydrochloric  acid,  0. 01  M 2, 967 

Sodium  hydroxid,  0.01  M 2, 756 

The  analysis  of  the  washed  and  dried  material  showed  that  it  con- 
tained 68  per  cent  carbohydrates,  18  per  cent  gelatine-like  material, 
and  14  per  cent  of  salts.  It  is  of  interest  to  note  that  these  algse,  which 
inhabit  the  shore,  display  a  course  of  acidity  through  the  day  generally 
similar  to  that  of  other  thick  and  succulent  plants  by  which  the  acidity 
is  highest  in  the  morning,  decreasing  toward  the  end  of  the  day,  but 
sometimes  rising  before  night.1 

The  vacuolar  fluid  of  the  plant-cell  may  be  taken  to  carry  minute 
quantities  of  the  carbohydrates  which  enter  into  the  protoplastic  gel 
at  all  times  and,  in  addition,  the  sugars  which  figure  so  prominently  in 
the  metabolism  of  the  plant.  The  mucilage  and  pentosans  in  general 
change  but  slowly  and  are  to  be  considered  as  being  of  importance 
chiefly  by  reason  of  their  properties  and  effects  as  constituents  of  the 
colloidal  structure.  The  presence  of  sucrose  and  dextrose  of  course 
modifies  the  osmotic  properties  of  the  cell,  and  as  these  substances  are 

1  Clark,  Lois.     Acidity  of  marine  algse.     Puget  Sound  Marine  Sta.  Publ.  1,  No.  22.     1917. 


Constituents  of  Biocolloids  Affecting  Hydration  and  Growth.  29 

dissolved  in  the  fluids  which  are  imbibed  by  the  gels,  their  probable 
influence  is  a  matter  of  some  importance.  An  examination  of  the 
effects  of  sucrose  and  dextrose  on  agar  and  gelatine  and  mixtures 
of  these  two  substances  was  made  by  Dr.  E.  E.  Free  at  the  Coastal 
Laboratory,  Carmel,  California,  in  September  1916,  and  his  results 
show  no  certain  effect  upon  the  hydration  of  gelatine,  agar,  and  of 
mixtures  of  the  two  substances  from  water  solutions  containing  as  much 
as  25  per  cent  of  sucrose  or  dextrose.1  More  recently,  E.  A.  and  H.  T. 
Graham  have  found  that  glucose,  saccharose,  and  lactose,  when  added 
to  gelatine,  retard  the  diffusion  of  such  acids  as  hydrochloric,  nitric, 
sulphuric,  phosphoric,  lactic,  formic,  acetic,  and  butyric,  with  an  accom- 
panying effect  on  the  swelling.  Diffusion  was  also  found  to  be  retarded 
by  sodium  chloride.2  The  universal  presence  of  both  sugars  and  salts 
in  the  plant  cell  gives  great  importance  to  the  relations  indicated. 

Mucilages  or  pentosans  are  present  in  varying  proportions  in  all 
plant  cells,  and  it  is  the  character  and  relative  amounts  of  such  com- 
pounds that  largely  determine  the  hydration  reactions  of  the  proto- 
plast. Of  these  substances  agar  was  used  most  generally  throughout 
the  experiments,  because  it  goes  into  the  disperse  condition  very  slowly 
and  in  this  particular  is  identical  with  protoplasmic  gels. 

According  to  information  furnished  by  Mr.  H.  Nakano,  of  the  Botan- 
ical Garden  of  Tokyo,  agar  is  prepared  chiefly  from  the  algae  Gelidium 
amansii  Lamour,  G.  pacificum  Okam,  G.  linoides  Kiitz,  Pterocladia 
capillacea  Born,  et  Thur.,  while  some  material  of  Gelidium  subcostatum 
Kiitz,  Ceramium  boydenii  Gepp.,  Campylcephora  hypenaloides  Y.  Ag., 
Acanthopeltis  japonica  Okam,  etc.,  may  be  included.  The  process 
includes  washing  in  fresh  water,  decoloration  in  the  sun,  milling,  boil- 
ing, filtering,  maceration  in  sulphuric  or  acetic  acid,  freezing,  and 
drying.  Modernized  methods  simplify  this  treatment  somewhat. 
Salts  and  nitrogen  are  present  in  the  final  product  in  minute  quan- 
tities insufficient  to  affect  hydration.3 

Other  gums  and  mucilages  of  this  group  which  were  tested  included 
gum  arabic  or  acacia,  cherry  gum,  prosopis  gum,  tragacanth,  and 
opuntia  mucilage,  all  of  which  are  more  readily  dispersible  in  water, 
but  which  do  not  go  wholly  into  suspension  even  in  prolonged  im- 
mersion. The  mucilages  of  the  cacti  are  pentosans,  or  substances 
which  yield  hexose  and  pentose  sugars  on  hydrolysis  with  dilute  acids. 
Substances  of  a  similar  character  formed  by  the  condensation  from 
simpler  sugars  may  be  taken  to  be  universally  present  in  plant  cells, 
being  aggregated  in  the  plasmatic  mesh,  in  which  condition  the  muci- 

1  Free,  E.  E.     Note  on  the  swelling  of  gelatine  and  agar  gels  in  solutions  of  sucrose  and  dextrose . 

Science,  46  :  142.     1917. 

2  Graham,  E.  A.  and  H.  T.     Retardation  by  sugars  by  diffusion  of  acids  in  gels.     Jour.  Amer . 

Chem.  Soc.  40  :  1900.     1918. 

3  For  further    information  concerning  the  origin  and  preparation  of  similar  products,   see 

Swartz,  M.  D.     Nutrition  investigations  on  the  carbohydrates   of  lichens,  algse,  and  re- 
lated substances.     Trans.  Conn.  Acad.  of  Arts  and  Sciences,  16:247-382.     Apr.  1911. 


30 


Hydration  and  Growth. 


lages  and  gums  elude  microchemical  tests.  It  has  already  been  pointed 
out  that  it  is  in  this  condition  that  they  produce  the  peculiar  hydration 
properties  of  living  matter  which  are  those  of  an  agar-protein  gel.1 

Generally  the  mucilages  originate  in  minute  quantities  in  numerous 
places  in  the  protoplasm,  but  when  such  structures  as  starch-grains  or 
layers  of  wall  material  are  transformed,  the  gels  so  formed  largely  re- 
mains in  place,  and  as  they  swell  to  occupy  a  much  larger  space  than 

TABLE  11. — Hydration  of  sections  containing  gums  and  mucilages. 


Distilled 
water. 

Citric 
acid, 
0.01  N. 

Sodium 
hydroxid, 
0.01  M. 

Potassium 
nitrate, 
0.01  M. 

Agar  (17°  C.)  

p.  ct. 
2,420 

1,760 
fl  ,141 
\1,072 
/     750 
\1,278 
1,417 
2,020 
1,684 
2,178 
/     846 
\1,038 
900 

p.  ct. 
1,300 

1,182 
957 
572 
889 
912 
1,050 
1,100 
947 
1,367 
1,500 
1,500 
650 

p.  ct. 
602 

824 
478 
458 
639 
556 
750 
520 
474 
778 
731 
731 
350 

p.  ct. 
1,700 

Agar  6,  prosopis  gum  2,  gel.  1,  bean 
protein  1  (25°  C.)  

Agar  6,  gum  arabic  3,  gel.  1  (14- 
17°  C.)  

1,250 
1,389 
1,082 
1,100 
1,268 
1,347 
1,725 
1,346 
1,200 
300 

Agar  8,  cherry  gum  2  (16°  C.)  

Agar  6,  cherry  gum  3,  gel.  1  (16°  C.). 
Agai  8,  cherry  gum  (precip.),  2.  ... 
Agar  8,  gel.  2  (16°  C.)  

Agar  8,  tragacanth  2  (15°  C.)  

Tragacanth  (15°  C.)  

Opuntia  mucilage  (15°  C.)  

Water. 

Hydrochloric 
acid, 
0.01M. 

Sodium 
hydroxid, 
0.01M. 

Agar  6,  opuntia  mucilage  2,  bean  protein  1, 
gel.  1  (26-27°  C.)  

p.  ct. 
1,780 
2,400 

500 
425 
400 
387 

p.  ct. 
780 

900 
806 
762 
806 
706 

p.  ct. 
1,060 

950 
562 
531 
575 
612 

(22°  C.)  

Gelatine  90,  cactus  mucilage  10  

Average  

428 

329 
431 

770 

850 
789 

557 

685 
431 

Gelatine  100,  agar  5,  averages  

Gelatine  80,  agar  20,  averages  

that  occupied  by  the  bodies  from  which  they  were  formed,  the  resultant 
masses  may  be  so  large  as  to  crowd  the  protoplasm  into  a  small  com- 
pass.2 Their  hydration  offers  such  indeterminate  features  as  to  make 

1Spoehr,  H.  A.    Carbohydrate   economy  of   the   cacti.     Carnegie  Inst.    Wash.  Pub.   No.  287 

pp.  44-47.     1919. 
2Stewart,  E.  G.   Mucilage  or   slime   formation   in  the    cacti.     Bull.  Torr.  Bot.  Club  46: 175. 

1919.     Lloyd,  F.  E.     The  origin  and  nature  of  the  mucilage  in  the  cacti  and  in  certain 

other  plants.     Amer.  Jour,  of  Bot.,  6:156.     1919. 


Constituents  of  Biocolloids  Affecting  Hydration  and  Growth.  31 

it  impossible  to  secure  measurements  by  the  methods  which  may  be 
used  with  agar  and  with  gelatine.  Information  as  to  their  effects  can 
only  be  obtained  by  observations  on  the  action  of  mixtures  of  which 
they  form  a  part.  Table  11  includes  some  of  the  data  as  to  the  swell- 
ing of  colloids,  including  pentosans  secured  in  this  laboratory. 

It  is  also  obvious  that  the  addition  of  any  of  these  gums  or  mucilages 
to  agar  tends  to  lessen  swelling  in  water  and  to  equalize  the  imbibition 
in  water  and  in  acids.  Their  general  effect,  however,  when  combined 
with  nitrogenous  substances,  is  to  make  a  colloid  which  has  a  higher 
coefficient  of  swelling  in  water  than  in  organic  acids,  although,  as  may 
be  seen  later,  a  special  relation  is  sustained  to  the  amino-acids. 

The  vacuolar  fluid  of  the  plant  cell  probably  always  contains  some 
protein  or  its  derivatives  in  the  form  of  amino-acids,  while  various 
nitrogenous  compounds  have  been  identified  in  the  nucleus  and  other 
bodies  of  morphological  rank.  The  formation,  disintegration,  and 
migration  of  these  substances  from  one  part  of  the  cell  to  another 
offers  a  most  inviting  field  for  the  researcher  concerned  with  the  physics 
of  the  cell. 

The  proteinaceous  substances  are  of  course  invariable  constituents 
of  the  biocolloids  of  the  plant  protoplast.  The  varying  reactions  of 
such  material  to  the  hydrogen-ion  concentration  or  acidity  of  solu- 
tions and  to  salts  are  exemplified  in  nearly  every  section  of  this  work. 
Combinations  of  agar  with  protein  extracts,  with  albumins,  peptones, 
gelatine,  and  amino-acids  were  tested  to  such  an  extent  that  it  is 
possible  to  say  that  the  highest  coefficients  of  hydration  in  water  alone 
are  exhibited  by  pentosan-albumin  mixtures  in  which  the  substances 
of  the  first  group  form  the  greater  part  of  the  mixture.  All  such  trials 
were  with  materials  with  possible  physiological  significance,  especially 
in  plants. 

Many  of  the  nitrogenous  compounds  used  in  making  biocolloids  in 
our  tests  are  known  to  be  actually  present  in  the  cell.  The  presence 
of  one  of  them,  peptone,  in  the  nucleus  is  definitely  established.  A 
characteristic  behavior  of  the  mixtures  containing  such  substances  has 
already  been  noted  (see  MacDougal  and  Spoehr,  The  effects  of  acids 
and  salts  on  biocolloids,  Science,  46:  269.  1917).  Increases  of  nearly 
3,200  per  cent  in  distilled  water,  567  per  cent  in  hundredth-molar 
hydrochloric  acid,  and  the  superior  and  long-continued  swelling  in 
hundredth-molar  potassium  hydroxid,  which  sometimes  reached  a 
total  of  nearly  1,700  per  cent  at  room  temperatures  (20°  to  28° C.),  were 
the  characteristic  features.  These  figures  were  obtained  by  the  use  of 
sections  consisting  of  90  parts  agar  and  10  parts  Witte's  peptone. 

The  tests  were  repeated,  using  "Diffco"  peptone  in  the  same  pro- 
portion and  with  temperature  kept  strictly  at  15°,C.,  and  the  records 
given  below  are  all  at  the  close  of  22  hours.  The  measurements  were 
as  follows: 


32  Hydration  and  Growth. 

TABLE  12. 

p.  ct. 

Water 1,400 

Potassium  nitrate,  0.01  M 1 , 200 

Potassium  nitrate,  citric  acid,  0.01  N 900 

Citric  acid,  0.1  N 675 

Potassium  nitrate,  potassium  hydroxid,  0.01  M 575 

Potassium  hydroxid,  0.01  M 400 

The  hydration  in  all  the  solutions  was  less  than  in  the  earlier  tests, 
partly  due,  no  doubt,  to  the  difference  in  the  peptones  used.  The 
increase  in  hydroxid  is  small;  no  direct  comparison  can  be  made  as  to 
the  acid,  as  a  hundredth-normal  solution  was  used  in  the  other  tests, 
while  the  swelling  in  acidified  salt  solutions  is  relatively  large.  Such 
results  emphasize  the  fact  that  material  standardized  for  cultural 
and  chemical  purposes  may  present  differences  in  colloidal  action  of  a 
serious  character. 

Nucleinic  acid  is  a  substance  of  interest  in  connection  with  its 
occurrence  in  the  nucleus  and  its  direct  effect  in  simple  combination 
was  tested.  A  mixture  of  90  parts  agar  and  10  parts  nucleinic  acid 
was  dried  into  plates  0.2  mm.  in  thickness.  Two  series  of  sections 
were  swelled  in  a  dark  room  at  16°  C.,  with  the  following  results: 

TABLE  13. 

p.  ct.  p.  ct. 

Water 1,400  1 ,025 

Potassium  nitrate,  0.01  N 900  800 

Potassium  nitrate,  citric  acid,  0.01  N 650  675 

Potassium  nitrate,  0.01  N 850  750 

Potassium  citrate,  citric  acid,  0.01  N 725  .... 

Citric  acid,  0.  01  N, 700  625 

Sodium  hydroxid,  0.01  M 1,000  925 

The  action  of  this  mixture  fixes  attention  by  reason  of  the  extra- 
ordinarily high  amount  of  swelling  in  the  alkaline  solution.  The 
amount  of  swelling  in  acid  does  not  average  more  than  half  that  in 
distilled  water.  The  behavior  of  nucleinic  acid  and  of  peptone  when 
mixed  with  agar  is  such  as  to  suggest  that  a  study  of  the  action  of 
these  substances  when  combined  separately  and  together  with  agar 
would  be  of  interest  in  connection  with  interpretations  of  nuclear 
phenomena. 

The  series  of  tests  now  including  some  data  from  mixtures  of  most 
of  the  albumin  and  protein  derivatives  which  were  available,  it  was 
deemed  advisable  to  introduce  more  than  one  nitrogenous  compound 
into  the  biocolloid.  The  first  mixture  contained  90  parts  agar,  3  parts 
nucleinic  acid,  3  parts  peptone,  and  4  parts  of  asparagine.  The  plates 
dried  to  a  thickness  of  0.2  mm.  and  sections  were  swelled  in  a  room 
at  15°  C.  with  the  results  as  given  in  table  14. 

The  outstanding  features  in  table  14  are  the  comparatively  high 
amount  of  swelling  in  the  salts.  The  expected  high  hydration  in  acid 
solutions  is  not  exhibited. 


Constituents  of  Biocolloids  Affecting  Hydration  and  Growth. 


33 


TABLE  14. 


Water. 


p.  ct. 
1,025 


Potassium  nitrate,  0.0  M 950 

Potassium  nitrate,  citric  acid,  0.01  N 675 

Citric  acid,  0.01  N 650 

Potassium  nitrate,  potassium  hydroxid,  0.01  M 775 

Potassium  hydroxid,  0.01  M 575 

The  comparative  test  of  the  result  of  the  inclusion  of  aspartic  acid 
and  of  its  amine,  asparagine,  in  the  biocolloid  is  important,  because  the 
amine  is  a  noticeable  constituent  of  plant  cells,  in  which  it  is  frequently 
abundant.  The  acid  appears  to  be  only  sparingly  soluble  and  in  a 
plate  of  agar  90  parts  and  10  of  this  acid  aggregates  as  whitish  lumps  in 
the  plate  or  as  an  efflorescence  on  the  surface.  Much  of  the  last  form- 
ation comes  off,  so  that  the  proportions  given  above  do  not  hold  for  the 
dried  material.  The  asparagine  forms  clear  plates  with  the  agar. 
The  swellings  were  as  follows : 

TABLE  15. 


Citric 

Sodium 

Water. 

acid, 

hydroxid, 

0.01  N. 

0.01  M. 

p.  ct. 

p.  ct. 

p.  ct. 

Agar,  asparagine.  .  .  . 

640 

300 

402 

Agar,  aspartic  acid  . 

295.5 

250 

625 

The  asparagine  mixture  shows  swellings  in  water,  acids,  and  alkali 
not  widely  different  in  proportion  from  those  hi  which  proteids  of  the 
bean  are  used.  The  aspartic  acid,  in  accordance  with  expectation, 
shows  an  amplitude  of  swelling  characteristic  of  organic  acids  in  the 
solution  in  both  acid  and  distilled  water  as  reagents.  Neutralization 
by  hydroxid,  and  the  renewal  of  this  reagent,  was  followed  by  greater 
swellings. 

The  introduction  of  a  fat  into  a  biocolloid  was  attempted  with  a 
preparation  of  lecithin  (Merck)  from  eggs.  An  amount  which  would 
give  a  proportion  of  about  0.5  per  cent  was  smeared  as  a  coating  on  a 
glass  stirring-rod.  After  half  the  contents  of  a  flask  containing  a 
mixture  of  agar  90  parts  and  milk  albumin  9.5  parts  had  been  poured 
on  a  glass  plate  for  drying,  the  remainder  was  stirred  until  all  of  the 
lecithin  had  dissolved  from  the  rod.  It  was  then  poured  on  a  glass 
slab  to  cool.  Separate  particles  could  not  be  distinguished  with  a 
hand  lens,  but  the  mixture  had  a  brownish  tinge.  When  the  film  had 
dried  to  a  thickness  of  0.2  mm.  it  was  tested  under  auxographs  at  16° 
to  18°  C.  and  the  swelling  measurements  given  in  table  16  obtained. 

No  distinct  effect  of  the  fatty  substance  can  be  detected  in  these 
reactions,  nor  were  any  departures  discernible  in  another  preparation 
containing  90  parts  of  agar,  9  parts  of  bean  protein,  and  1  of  lecithin. 
The  bean  protein  is  a  water  extract  of  Phaseolus  vulgaris  containing 
the  albumins  and  also  the  other  proteins  soluble  in  the  salts  present. 


34 


Hydration  and  Growth. 


When  such  material  is  stirred  into  distilled  water  a  clear  solution  is 
obtained.  The  mixture  was  added  to  the  melted  agar  at  about  30°  C. 
An  amount  of  lecithin  (Merck)  from  eggs,  supposed  to  be  about  a  gram, 
was  smeared  on  the  outer  wall  of  a  thin  vial.  The  vial  was  dropped 
into  the  warm  mixture  and  shaken  until  it  had  nearly  all  passed  into 

TABLE  16. 


Water. 

Citric 
acid, 
0.01  N. 

Sodium 
hydroxid, 
0.01  N. 

p.  ct. 
1,923 
1,150 

p.  ct. 
423 
200 

p.  ct. 
250 
423 

Average  

1,536 

311 

336 

Averages  of  swelling 
of  mixture  lack- 
ing lecithin  

1,791 

333 

336 

the  solution,  giving  it  a  brownish  tinge.  By  another  method  the 
lecithin  was  smeared  on  the  inner  surface  of  a  warmed  flask.  The 
agar-protein  mixture  was  poured  in  at  a  temperature  of  about  45°  to 
50°  C.  and  shaken  until  all  of  the  lecithin  had  been  taken  up.  Dried 
plates  prepared  in  this  way  showed  no  important  departure  from  the 
behavior  of  mixture  without  lecithin.  A  series  of  such  swellings  with 
a  plate  0.47  mm.  in  thickness  at  16°  to  18°  C.  gave  the  following: 

TABLE  17. 

p.  ct. 

Water 1, 106 

Citric  acid,  0.01  N 329 

Sodium  hydroxid,  0.01  M 436 

It  is  obvious  that  these  crude  tests  by  no  means  constitute  an  ade- 
quate trial  of  the  effects  of  fats  or  lipins  in  hydration  of  living  matter. 
The  prominence  of  the  lipoid  theory  of  the  cell-membrane  and  the 
weight  of  some  of  the  arguments  adduced  in  its  support  renders  it 
highly  important  that  refined  methods  of  experimentation  be  used  in 
incorporating  a  lipin  colloid  in  pentosan-protein  mixtures,  the  hydration 
of  which  might  yield  results  of  importance  bearing  on  permeability. 

In  an  effort  to  make  a  mixture  bearing  a  closer  resemblance  to  the 
general  hydration  relations  of  plant  protoplasm,  the  following  mate- 
rials were  assembled: 

Agar  4.2  grams,  which  was  liquefied  in  160  c.c.  of  water  at  tempera- 
tures of  about  100°  C.,  oat  protein  0.18  gram,  and  oat  albumin  0.820 
gram,  were  dissolved  by  shaking  up  with  50  c.c.  cold  water.  After  this 
had  been  done  0.2  gram  of  lanolin  was  put  in  a  vessel  with  the  dis- 
solved albumin  and  warmed  to  about  35°  C.,  being  shaken  vigorously 
at  intervals.  The  agar  was  now  strained  through  two  layers  of  cheese 
cloth  into  a  beaker  and  stirred  until  it  came  down  to  a  temperature  of 
about  40°  C.,  when  it  was  placed  in  an  enameled  cup  suitable  for  the 


Constituents  of  Biocolloids  Affecting  Hydration  and  Growth.  35 

action  of  an  ordinary  revolving  egg-beater.  The  albumin-lanolin 
mixture  was  now  added  to  the  agar  and  it  was  stirred  vigorously  some 
time  above  35°  C.,  and  when  it  had  come  down  to  33°  C.  it  was  cast  in 
two  portions.  One  was  on  filter-paper  which  was  stretched  in  the 
usual  manner,  and  the  other  smaller  lot  on  a  glass  plate.  The  mixture 
set  in  a  few  minutes,  the  room  temperature  being  about  20°  C.  The 
plates  as  above  came  down  to  an  average  thickness  of  0.2  mm.,  which 
were  tested  at  a  temperature  of  18°  C.,  swelling  2,100  per  cent  in  dis- 
tilled water  and  but  1,750  per  cent  in  0.01  M  asparagine. 

A  conception  of  living  matter  as  simply  a  two-phase  colloid  in  which 
the  main  elements  are  distributed  between  the  more  liquid  and  the 
denser  phases  simply  according  to  their  physical  properties  may  be 
sufficient  to  interpret  certain  general  reactions,  but  one  does  not  pro- 
ceed very  far  with  the  actual  mechanics  of  living  matter  until  it  is 
realized  that  specializations  of  various  kinds  come  in.  Attention  has 
already  been  called  to  the  varying  proportion  of  proteins  and  their 
probable  effect  on  hydration.  According  to  Kite,1  the  vacuolar  fluid 
of  Spirogyra  contains  some  proteins  in  a  dissolved  or  disperse  state, 
and  this  fluid  is  even  higher  in  nitrogenous  material  in  Char  a.  A 
doubtful  amount  of  plasmatic  material  may  be  taken  to  be  hi  a  dis- 
perse condition  in  plants  under  ordinary  conditions,  and  the  heavier 
portions  differ  widely  as  to  density  or  viscosity,  the  outer  layer  of  the 
nucleus  and  of  the  cell  being  a  gel  of  greater  rigidity  than  the  interior 
portions.  The  fact  that  "none  of  the  cytoplasm  goes  into  solution 
very  readily  even  when  cut  into  very  minute  pieces,"  as  described  by 
Kite  with  respect  to  Spirogyra,  can  not  be  taken  to  prove  that  pro- 
toplasm may  not  readily  go  into  the  disperse  or  liquid  phase  in  the 
fluids  of  the  cell.  The  pentosans  may  move  slowly,  but  during  the 
growth  of  the  nucleus,  water  and  other  substances  pass  into  it  from  the 
surrounding  cytoplasm.  Again,  at  other  times,  material  may  be  seen 
to  pass  from  the  nucleus  to  the  cytoplasm.  Many  of  these  phenomena 
may  now  be  explained  on  known  behavior  of  colloids,  and  the  study 
of  colloidal  action  promises  to  yield  much  additional  information  upon 
the  movement  and  interaction  of  the  parts  of  the  protoplast. 

Most  of  the  variations  in  composition  mentioned  are  illustrated  in 
the  structure  of  a  single  cell,  and  in  the  growth  and  development  of 
these  units  the  accumulation,  migration,  and  disintegration  of  these 
substances  may  be  definitely  connected  with  the  more  important 
movements  in  the  cell.  The  localization  of  salts  is  a  matter  which 
has  been  dealt  with  at  great  length  by  MacCallum,  and  the  results 
which  he  has  secured  with  a  few  of  the  more  important  salts  afford  a 
basis  for  some  conception  of  the  heterogeneity  of  the  cell  with  regard 
to  this  feature. 

1  Kite,  G.  L.  The  physical  properties  of  protoplasm  of  certain  plant  and  animal  cells.  Amer. 
Jour,  of  Physiol.,  32  : 146,  especially  pp.  161  and  162.  1913.  See  also  Conklin,  E.  G.  Effects 
of  centrifugal  force  on  the  structure  and  development  of  the  eggs  of  Crepidula.  Jour.  Exper. 
Zool.,  22:  Feb.  1917.  See  pp.  356-364. 


36  Hydration  and  Growth. 

Migrations  of  albuminous  material  from  one  part  of  the  cell  to 
another  and  translocation  of  the  proteins  is  a  subject  upon  which 
nearly  all  cytologists  speak  with  great  reserve  because  of  the  lack  of 
well-grounded  observations.  An  extensive  examination  of  such  action 
by  the  use  of  root-tips  of  yiciafaba  has  been  made  by  Professor  C.  F. 
Hottes,  of  the  University  of  Illinois,  and  some  of  his  results  as  yet 
unpublished  include  facts  of  great  possible  importance.  Dr.  Hottes 
says1  that  seedlings  deprived  of  the  cotyledons  and  grown  in  the  dark 
at  20°  C.,  being  supplied  with  nutrient  salts  and  water,  continued  to 
grow  for  a  period  of  three  days  to  a  week.  The  changes  in  the  nucleus 
and  the  concomitant  action  of  the  cytoplasm  during  this  time  are  very 
striking  in  the  root-tips.  The  nucleolus  is  enormously  reduced  in  size 
and  its  materials  escape  into  the  cytoplasm.  Materials  from  distant 
cells,  albuminous  in  nature,  are  transported  through  considerable 
distance  to  the  meristem  of  the  tip,  and  these  cells  remain  alive,  sustain- 
ing a  parasitic  relation  to  the  cells  from  which  the  material  has  been 
derived,  and  the  fundaments  of  lateral  roots  are  broken  down  and 
translocated  in  the  same  manner.  The  progress  of  the  translocation 
may  be  followed  through  the  strands  connecting  with  the  tip  meristem. 
Such  transfer  of  materials  is  apparently  inhibited  at  low  and  high 
temperatures  which  lessen  or  stop  growth.  Antipyrine  accelerates 
exudation  and  transfer  of  such  proteinaceous  material  and  chloral 
hydrate  inhibits  it.  Furthermore: 

"In  all  treatments  leading  to  inhibition  of  cell  activity,  I  find  enlargements 
of  nucleolus,  increase  of  chromatin  without  the  passage  of  perceptible  amounts 
of  these  materials  into  the  cytoplasm.  In  cell  acceleration  the  nucleolar 
material  can  be  distinctly  followed  through  the  reticulum  of  the  nucleus  into 
the  cytoplasm.  The  chromatin  (tropochromata)  fluctuates  in  quantity  and 
its  increase  and  decrease  is  concomitant  with  the  absence  and  presence  of 
chromatin  (chromidia)  in  the  cytoplasm." 

As  the  proteins  diffuse  sparingly,2  their  translocation  in  living  mat- 
ter must  take  place  by  some  other  method,  and  one  by  which  a  rela- 
tively rapid  movement  would  be  possible.  So  far  as  plants  are  con- 
cerned, the  possibilities  offered  by  the  amino-acids  may  prove  to  be 
of  the  greatest  importance  in  this  connection.  These  substances  pass 
through  membranes,  show  a  relatively  high  rate  of  diffusion,  and  are 
readily  derived  and  combined. 

1  Letter  to  author. 

8  Robertson,  T.  B.     The  physical  chemistry  of  proteins.     New  York.     1918.     See  p.  330. 


IV.  THE  EFFECT  OF  SALTS  AND  ACIDS  ON  BIOCOLLOIDS 
AND  CELL-MASSES. 

A  proper  supply  of  certain  salts  in  the  substratum  is  one  of  the  most 
important  requirements  of  the  plant,  and  those  known  to  the  physiolo- 
gist as  necessary  for  growth  and  development  are  designated  as  "nutri- 
ent salts,"  although  more  properly  to  be  designated  as  culture  salts. 

Available  analyses  show  the  general  proportion  of  the  various  sub- 
stances present  in  the  organs  and  tissues  of  many  kinds  of  plants.  The 
specialized  or  localized  accumulations  in  the  regions  of  the  cell  have 
been  demonstrated  of  only  a  very  few  substances,  of  which  iron  and 
potassium  seem  to  be  the  most  notable.1  Chemical  unions  or  precipi- 
tations may  account  for  the  local  concentration  in  some  cases,  while  in 
other  structures  the  surface  tensions  of  the  minute  masses  of  gels  or 
liquid  may  be  responsible. 

The  heterogeneous  character  of  living  matter  and  the  known  facts 
of  its  hydration  and  that  of  biocolloids  by  which  water,  acids,  and 
salts,  etc.,  enter  into  combination  in  both  definite  and  indefinite  pro- 
portions with  the  colloidal  material,  together  with  the  behavior  of 
cell-masses  in  imbibition,  have  made  it  seem  inadvisable  to  attempt  to 
express  the  reactions  obtained  in  terms  of  hydrogen-ion  or  hydroxyl- 
ion  concentrations,  and  but  few  measurements  of  this  kind  are  cited  in 
the  experiments  described  in  the  present  paper.  Although  this  method 
entails  a  treatment  empirical  to  a  certain  extent,  yet  forced  parallel- 
isms and  false  explanations  resulting  from  the  application  of  simple 
formulae  to  complex  phenomena  are  avoided.  Attention  has  been 
confined  chiefly  to  the  study  of  the  action  of  solutions  in  which  dis- 
sociation may  be  assumed  to  be  complete  or  nearly  so.  By  following 
this  simplified  procedure  it  has  been  possible  to  explore  wide  fields  of 
biological  possibilities,  the  exact  mapping  of  which  will  need  con- 
centrated attention  upon  comparatively  narrow  problems.  This  is 
especially  true  of  the  action  of  the  amino-compounds  upon  biocolloids, 
concerning  which  certain  preliminary  results  are  described  in  the  fol- 
lowing pages. 

The  amount  of  acid  or  salts  and  of  water  which  may  be  taken  up 
from  a  solution  and  the  accompanying  swelling  is  influenced  by  several 
factors.  The  reader  is  referred  to  texts  on  physics  and  on  colloids  for 
detailed  discussions  of  adsorption  equations  and  for  information  con- 
cerning the  allowable  generalizations  concerning  the  relative  amounts 
of  material  which  may  be  taken  up  by  a  colloid  from  a  solution  system. 

For  the  present  some  results  recently  obtained  by  Miss  C.  L.  Carey 
and  as  yet  unpublished  will  be  of  interest,  as  the  absorption  of  water 

1  MacCallum,  A.  M.  The  distribution  of  potassium  in  animal  and  vegetable  cells.  Jour,  of 
Physiol.,  32  :95.  1905.  Also,  Presidential  address,  Brit.  Assoc.  Adv.  Sc.,  Report  for  1910,  p.  744. 

37 


38 


Hydration  and  Growth. 


and  hydrochloric  acid  taken  from  a  solution  by  various  materials  comes 
within  the  range  of  this  chapter.  In  these  tests  3  to  6  grams  of  the 
colloid  or  of  the  plant  material  were  placed  in  a  dish  containing  about 
100  c.  c.  of  the  acid  solution  at  21°  C.  The  results  are  given  in  table  18. 

TABLE  18. — Water  absorbed  from  hydrochloric  acid  per  gram  dry  substance. 


Material. 

Concentration. 

N/20. 

N/10. 

N/5. 

N/2. 

Agar  A                  

2.312 
4.242 

2.164 

Agar  B                      

3.497 
3.570 

3.571 
3.461 

Agar  C  (only  one  determination  in  each 
cone,  for  agar  C)        

4.275 

3.917 
2.944 

Agar  A  (48  hours)                 

Agar  A  (4  days)                             

3.693 

Agar  A  (7  days)  

2.688 

Agar  A  (10  days)  ...  .1.  

2.678 

Agar  A  (14  days)  

2.731 

Agar-gelatin  

4.520 
14.362 
1.368 
1.096 
1.169 
.822 

5.884 

4.065 
9.830 
1.504 
1.163 
1.181 
.804 

5.891 

3.821 
5.892 
1.514 
1.112 
1.257 
.786 

5.645 

3.913 
5.019 
1.602 
1.079 
1.210 
.812 

5.678 

Gelatine  (Cox's)    

Lupinus  albus  cotyledens  

Vicia  faba  cotyledons    

Phaseolus  lunatus  cotyledons    '.  .  . 

Starch  (commercial  "corn  starch").  .  .  . 
Coconut   (commercial   shredded,   after 
removal  of  oil  and  sugar)  

The  amount  of  acid  absorbed  was  greatest  in  all  cases  from  the  high- 
est concentrations  used.  The  amount  of  hydration  which  accom- 
panied the  incorporation  of  the  acids  in  the  colloids  is  given  in 
table  19. 

TABLE  19. — Absorption  of  hydrochloric  acid  and  water  from  solution,  per  gram  dry 

substance. 


Material. 

Hydrochloric  acid,  grams. 

Water,  grama. 

N/20. 

N/10. 

N/5. 

N/2. 

N/20. 

N/10. 

N/5. 

N/2. 

Agar  A             

0.01115 
.01177 

.01331 

0.01591 

2.312 
4.242 

4.275 

2.164 

3.917 
2.944 
3.693 
2  688 

Agar  B              

0.03168 
.03457 

0.06990 
.06892 

3.497 
3.570 

3.571 
3.461 

Agar  C  (only  one  determination 
in    each    concentration    for 
agar  C)      

.02151 
.01735 
.01913 
.01476 
.01386 

.01373 
.02508 
.05577 
.01700 
.01918 
.01996 
.00442 

.03585 

Agar  A  (48  hours),  4  determi- 
nations                         

Agar  A  (4  days),  2  determina- 
tions         .  .                    

Agar  A  (7  days),  3  determina- 
tions   

Agar  A  (10  days),  3  determina- 
tions 

2.678 

2.731 
4.065 
9.830 
1.504 
1.163 
1.181 
0.804 

5.891 

Agar  A  (14  days),  1  determina- 
tion   

.03433 
.06991 
.  02490 
.  02636 
.02672 
.00779 

.04789 

.07705 
.11075 
.04173 
.03191 
.03482 
.01434 

.08969 

4.520 
14.362 
1.368 
1.096 
1.169 
0.822 

5.884 

3.821 
5.892 
1.514 
1.112 
1.257 
0.786 

5.645 

3.913 
5.019 
1.602 
1.079 
1.210 
0.812 

5.678 

Agar-gelatine  

.01941 
.04130 
.00843 
.  01269 
.01256 
.00259 

.02344 

Gelatine  (Coxe's)  

Lupinus  albus  cotyledons  .  .  . 

Vicea  Faba  cotyledons  

Phaseolus  lunatus  cotyledans  .  . 
Starch  (commercial  starch)  .... 
Coconut  (commercial  shredded 
after  removal  of  sugar  and  oil)  . 

Effect  of  Salts  and  Acids  on  Biocolloids  and  Cell-masses. 


39 


Briefly  summarized,  agar  takes  up  the  greatest  amount  of  water  in 
24  hours  from  a  0.05  N  solution,  and  the  maximum  imbibition  in 
gelatine  and  gelatine-agar  combinations  also  ensues  in  this  concentra- 
tion, which  is  one  duplicated  in  the  cell-masses  of  the  plant.  Cotyl- 
edons and  sections  of  the  plants  tested  found  their  maximum  at  a 
concentration  of  0.1  N  at  the  temperatures  named. 

Hydration  of  dried  plates,  or  of  sections  of  living  plants,  is,  of  course, 
accompanied  by  a  diffusion  or  solution  out  of  the  contained  salts  in  a 
manner  determined  by  a  large  number  of  environmental  conditions, 
inclusive  of  the  proportionate  amount  of  water  to  which  the  colloid  is 
exposed  or  in  which  it  may  be  immersed. 

Thus,  in  most  of  the  experiments  described  in  this  volume,  the  sec- 
tions having  a  total  initial  volume  of  dried  material  of  about  2  or  3 
c.  mm.  were  immersed  in  dishes  containing  33  c.  c.  of  water.  The 
hydration  of  material  over  a  period  of  24  to  50  hours  would  necessarily 
result  in  the  solution  out  of  a  portion  of  the  salt  contained,  which  might 
form  as  much  as  18  per  cent  of  the  original  dried  weight  of  the  sec- 
tions. On  the  other  hand,  swelling  in  a  solution  of  salt-free  colloid, 
for  example,  might  result  in  an  accumulation. 

A  series  of  tests  was  made  to  ascertain  the  relative  amounts  of  water 
which  might  be  taken  up  by  one  of  the  biocolloids  used  extensively  in 
this  work  from  a  graded  series  of  a  salt  solu- 
tion. Since  it  showed  a  maximum  hydration 
capacity  at  temperatures  of  15°  to  40°  C.,  a  mix- 
ture of  agar  90  parts  and  oat  protein  10  parts 
was  used,  and  the  sections,  which  ranged  from 
0.16  to  0.18  mm.  in  thickness,  were  measured 
as  to  each  set  and  arranged  in  trios  in  glass 
dishes.  The  sections  were  as  nearly  uniform  as 
possible  and  the  average  volume  of  the  air-dry 
trios  of  sections  in  each  dish  was  12  c.  mm. 
The  testing  dishes  held  30  c.  c.  of  the  salt  solu- 
tion. Temperatures  were  taken  by  means  of 
small  thermometers  of  the  clinical  type,  and 
readings  of  the  temperature  of  the  solution  in 
the  dishes  were  made  several  times  during  the 
course  of  the  test.  It  is  to  be  noted  that  the 
end-point  of  the  swellings  would  not  have  been 
reached  until  after  40  to  48  hours  in  the  less- 
concentrated  solutions,  but  the  amount  of  ex- 
pansion which  might  have  been  displayed  in 

the  last  12  hours  of  this  period  would  not  have  changed  the  totals 
greatly  or  the  proportions  in  any  important  manner.  The  data  given 
in  table  20  represent  the  average  expansion  of  sets  of  3  sections  at 
16°  to  17°  C. 


TABLE  20. — Swelling  of  a 
mixture  of  agar  90  parts 
and  oat  protein  10  parts 
in  distilled  water  and 
potassium  nitrate  at  16° 
to  17°  C. 


Potassium  nitrate. 

2  M  

p.    ct 
445 
640 
530 
695 
860 
1,165 
1,305 
1,560 
1,670 
1,500 
1,670 
1,720 

1  M  

0.5  M  

.1  M  

.05  M  

.02  M  

.01  M  

.  005  M  

.  0025  M  

.00125  M  
.000625  M  
.0003125  M... 

Distilled  water 

1,940 

40 


Hydration  and  Growth. 


TABLE  21. 


The  range  of  concentrations  examined  does  not  exhaust  the  possi- 
bilities. It  has  been  previously  found  that  biocolloids  will  take  up 
some  water  and  swell  from  the  most  concentrated  mixtures  of  salts. 
On  the  other  hand,  the  greatest  attenuations  exerted  some  influence 
on  the  wTater  capacity,  although  it  may  be  surmised  that  at  a  lower 
concentration  the  deviation  on  the  swelling  in  the  salt  solution  from 
that  in  distilled  water  would  be  so  slight  as  to  be  negligible  in  all 
biological  applications  of  the  facts.  Living  matter  is  at  all  times 
impregnated  with  salts  to  a  degree  within  the  range  of  these  tests. 

In  the  continuance  of  the  series  to  test  the 
effects  of  some  of  the  salts  of  biological  im- 
portance, plates  of  agar  and  oat  protein  0.2 
mm.  in  thickness  were  swelled  at  15°  C.  in  cal- 
cium nitrate.  Two  series  of  measurements  are 
shown  in  table  21. 

The  weakest  attenuation  used  allows  a  swell- 
ing practically  equivalent  to  that  of  distilled 
water.  Two  series  of  greatest  divergence 
from  the  effects  of  potassium  nitrate  are  to  be  found  in  dilutions  of 
0.02  M,  while  distinctly  different  action  is  seen  to  prevail  in  the  con- 
centrated solutions  above  the  unimolecular. 

The  sections  of  agar-oat  protein  used  for  testing  the  effects  of  cal- 
cium chloride  were  0.16  mm.  in  thickness  and  the  swelling  was  made 
at  the  same  temperature  as  in  potassium  nitrate,  15°  C.  The  measure- 
ments in  24  hours  are  given  in  table  22. 

TABLE  22.  TABLE  23. 


Calcium  nitrate. 

p.  ct. 

p.  ct. 

2  M  

975 

917 

0.2  M.  .  . 

525 

722 

.02  M.. 

650 

778 

.002  M. 

1,425 

1,555 

.0002  M 

1,975 

Calcium  chloride. 

Distilled  water. 
2  M  

p.  ct. 
1,660 
273 
375 
656 
907 
1,279 
1,281 
1,469 
1,438 

p.  ct. 

1  M  

0.1  M  

.01  M  

.  005  M  

.001  M  

.0002  M  
.00005  M  

1,438 
1,688 

Potassium  chloride. 

Water. 

2  M  

p.  ct. 
468 
656 
1,031 
1,344 
1,156 
1,906 
2,210 
1,406 

p.  ct. 

p.  ct. 

p.  ct. 
1,781 
1,989 

1  M  

0.1  M  
.01  M  
.005  M... 
.001  M... 
.0002  M.. 
.00005  M. 

1,463 
1,625 
1,719 

1,467 
1,667 
1,533 

The  chloride  of  calcium  appears  to  limit  swelling  to  a  greater 
extent  than  the  nitrate,  so  far  as  table  22  is  comparable  with  that 
obtained  from  swelling  in  the  nitrate. 

The  next  trial  was  made  with  potassium  chloride  in  solutions  of  the 
same  concentration  as  above.  Agar-oat  protein  was  used  and  the 
swellings  at  15°  C.  in  24  hours  are  given  in  table  23. 

The  amount  of  imbibition  in  potassium  chloride  is  greater  than  that 
in  calcium  chloride  in  equivalent  concentrations,  while  it  is  noticeable 


Effect  of  Salts  and  Acids  on  Biocolloids  and  Cell-masses.  41 

that  in  the  0.0002  M  solutions  here,  as  in  some  of  the  trials  previously 
made,  the  swelling  is  very  high,  probably  even  higher  than  that  in  dis- 
tilled water. 

Swellings  of  the  agar-oat-protein  mixture  for  24  hours  at  15°  C.  gave 
the  results  shown  in  table  24  in  di-potassic  phosphate  (K^HPC^) . 

The  sections  in  the  1  M  solution  were  sealed 
by  the  glass  triangle,  which  was  pressed  too 
closely  on  them,  with  the  result  that  swelling 
progressed  very  slowly  to  a  defective  total. 


Di-potassic  phosphate. 


Distilled  water .  . 

2  M 

1  M 

0.1  M 

.01  M 

.005  M 

.001  M 

.0002  M 

00005  M.. 


p.  ct. 


156 

125 

625 

806 

1,563 

1,719 

1,889 

1,964 


If  the  results  of  the  swelling  in  the  di-potassic 
phosphate  were  plotted  as  a  graph,  it  would  be 
seen  that  the  steepest  part  of  the  curve  would 
lie  in  the  region  between  the  concentrations  of 
0.01  M  and  0.005  M.  The  graph  of  the  potas- 
sium chloride  would  be  a  much  more  regular 
figure.  The  steepest  part  of  the  graph  of  the 
results  with  calcium  chloride  would  probably 
lie  between  0.01  N  and  0.005  N,  the  steepest 
part  of  the  graph  of  calcium  nitrate  would  probably  be  in  the  region 
between  0.02  N  and  0.002  N,  and  the  steepest  part  of  the  line  express- 
ing the  falling-off  of  the  retarding  action  of  potassium  nitrate  would 
be  between  0.05  N  and  0.005  N. 

The  breaks  or  discontinuities  in  the  rise  of  the  curve  of  imbibition 
total  led  to  the  belief  that  some  errors  had  crept  in,  and  repetitions 
were  made  with  concentrations  from  0.01  M  to  0.000005  M.  The 
additional  experiments  were  for  the  most  part  symmetrical  with  each 
other,  although  it  is  not  allowable  to  contrast  the  separate  items  of  the 
swellings  of  two  different  lots  of  material.  The  ground  at  first  taken, 
that  in  minute  quantities  some  of  these  salts  might  cause  a  swelling  in 
excess  over  that  in  distilled  water,  still  lacks  confirmation.  It  is  a 
matter,  however,  that  should  be  tested  with  great  care,  as  such  re- 
actions for  sections  of  plants  are  included  in  my  records. 

The  swelling  in  various  concentrations  of  a  salt  supposedly  depends 
chiefly  upon  the  acid  ion,  although  the  action  of  the  basic  ions  is  not 
actually  excluded.  The  above  tests  were  made  in  solutions  varying 
from  4  M  to  0.5  M,  which  are  far  too  concentrated  to  be  of  direct 
biological  interest.  A  more  dilute  series  of  potassium  salts  in  0.01  M 
and  0.001  M  solutions  was  made  up  and  the  swelling  of  sections  of 
agar  90  parts  and  peptone  10  parts,  0.22  mm.  in  thickness,  were  made 
at  15°  C.  The  tests  were  closed  at  the  end  of  24  hours,  and  although 
some  slight  increase  was  still  in  progress,  the  relations  of  the  various 
preparations  were  identical  with  those  which  might  be  expected  of 
the  end-points.  (Table  25.) 

According  to  Hofmeister,  as  cited  by  Taylor,  swellings  of  gelatine 
in  chlorides  and  nitrates  should  be  greater  than  in  the  citrates  and 


42 


Hydration  and  Growth. 


TABLE  25. 


sulphates.  The  differences  found  in  my  own  tests  with  the  above 
mixtures,  which,  it  must  be  pointed  out,  are  so  small  as  to  be  very  close 
to  the  limit  of  variation,  are  of  the  reverse  kind.  They  are,  however, 
of  such  a  nature  as  to  warrant  the  assertion  that  the  greatest  swelling 
of  this  biocolloid  in  the  group  of  substances 
named  does  not  take  place  in  the  nitrates 
and  chlorides. 

Another  aspect  of  this  matter  was  tested 
by  arranging  a  series  in  which  ,an  agar- 
peptone  mixture  was  swelled  in  two  con- 
centrations of  sodium  acetate,  which  is  re- 
puted to  retard  imbibition  in  simple  col- 
loids, and  sodium  chloride,  which  is  said 
to  increase  the  amount  of  swelling  over 
that  of  water.  The  measurements  of  such 
swellings  at  15°  C.,  closed  at  the  end  of  24  hours,  are  given  in  table  26. 

These  results  are  featureless,  so  far  as  the  above  point  is  concerned. 
The  lesser  concentration  of  the  sodium  acetate  seems  to  give  a 
greater  swelling  than  the  higher,  but,  on  the  other  hand,  the  sodium 
chloride,  which  should  promote  imbibition,  does  not  induce  swelling 
as  great  as  those  in  the  acetate.  The  sections  used  were  salt-free  and 
a  parallel  series  was  run,  using  dried  sections  taken  from  the  median 
layer  of  Opuntia  joints,  which  at  15°  C.  gave  the  measurements  shown 
in  table  27. 

TABLE  26.  TABLE  27. 


Concentration. 

0.01  M. 

0.001  M. 

p.  ct. 

p.  ct. 

Chloride  .  . 

818 

1,136 

Nitrate  .  .  . 

818 

1,205 

Phosphate 

932 

1,136 

Citrate  .  .  . 

977 

1,227 

Sulphate  .  . 

J975 

1,250 

Estimated  from  0.007  M. 


Concentration. 

0.01  M. 

0.001  M. 

Sodium  acetate.  . 
Sodium  chloride  . 

p.  ct. 
1,167 
1,214 

p.  ct. 
1,262 
1,214 

Concentration. 

0.01  M. 

0.001  M. 

Sodium  acetate.  . 
Sodium  chloride. 
Distilled  water.  .  . 

p.  ct. 
650 
613 
570 

p.  ct. 
613 

588 

The  swelling  in  both  salts  is  greater  at  the  higher  concentration,  and 
the  maximum  effect  may  lie  at  a  higher  point.  The  acetate  induces  a 
higher  hydration  effect  than  the  chloride.  The  plant  sections  are  of 
course  extremely  complex  as  to  chemical  composition,  although  their 
relations  to  water  are  taken  to  be  chiefly  determined  by  the  pentosan- 
protein  ratio,  and  are  modified  by  the  salts  already  present  and  by  the 
residual  acidity.  It  is  evident  that  gelatine  and  isinglass  do  not 
furnish  conditions  for  swelling  analogous  to  those  of  the  plant,  as  as- 
sumed by  so  many  writers,  since  the  results  given  above  do  not  coincide 
in  the  main  with  those  obtained  by  Hofmeister.1  As  has  been  pointed 
out  elsewhere  in  this  work,  the  similarity  of  action  of  the  plant  to  that 


1  Hofmeister,  F.     Die  Betheiligung  geloster  Stoffe  an  Quellungs-vorgange. 
Pathol.  u.  Pharm.,  27:395,  1890,  and  28:210,  238,  1891. 


Archiv.  f.  Exper. 


Effect  of  Salts  and  Adds  on  Biocolloids  and  Cell-masses.  43 

of  gelatine  and  of  the  proteins  and  their  derivatives  will  depend  chiefly 
upon  the  proportions  of  such  substances  in  the  living  cell-masses.  The 
properties  of  gelatine  may  illustrate  those  of  protoplasm  only  in  so  far 
as  they  are  general  to  the  elastic  gels,  in  which  class  of  colloids  both  may 
be  included.1 

The  salt-content  of  colloids  of  living  matter  in  all  probability  changes 
very  slowly,  while  the  acidity  may  vary  with  great  rapidity  and  through 
a  wide  range.  A  set  of  tests  were  therefore  arranged  in  which  the 
salt-content  would  remain  constant  while  the  solutions  contained  a 
series  of  acid  concentrations.  The  first  series  was  one  in  which  the 
salt  was  dissolved  in  the  solution  of  the  acid  after  the  manner  in  which 
many  measurements  have  been  previously  made.  Sections  of  plates 
of  agar  90  parts  and  oat  protein  10  parts  which  had  an  average  thick- 
ness of  0.18  mm.  were  cut  so  that  a  trio  had  a  total  volume  when  air- 
dry  of  12  cu.  mm.  and  the  dishes  in  which  these  were  placed  held  about 
30  c.  c.  of  the  solution.  These  measurements  were  made  with  the  solu- 
tions standing  at  16°  to  17°  C.  The  results  were  as  follows: 

TABLE  28. 

p.  ct. 

Distilled  water 1 , 722 

Potassium  nitrate,  0.01  M 1, 250 

Potassium  nitrate,  0.01  M  +  citric  acid,  0.05  N 472 

Potassium  nitrate,  0.01  M  +  citric  acid,  0.01  N 628 

Potassium  nitrate,  0.01  M  +  citric  acid,  0.005  N 667 

Potassium  nitrate,  0.01  M  -j-  citric  acid,  0.001  N 944 

Potassium  nitrate,  0.01  M  -J-  citric  acid,  0.0002  N 1,055 

Potassium  nitrate,  0.01  M  +  citric  acid,  0.00004N 1, 139 

The  above  measurements  were  taken  at  the  end  of  24  hours,  at 
which  time  the  sections  in  distilled  water  and  in  the  two  solutions 
containing  least  acid  were  still  slowly  expanding,  at  a  rate  which 
would  not  have  changed  the  final  aspect  of  the  test.  These  results 
are  of  importance,  since  it  has  been  found  that  the  range  of 
acidity  in  such  plants  as  growing  joints  of  cacti  may  be  practically 
equivalent  to  that  from  the  highest  acid-content  to  the  lowest  during 
the  daylight  period,  coincident  with  an  enormous  variation  in  the 
water-capacity  of  the  organ.2  The  measurements  of  plates  composed 
of  90  parts  agar  and  10  parts  bean  protein  0.25  mm.  in  thickness,  in 
dark  room,  at  16°  to  17°  C.,  gave  the  results  shown  in  table  29. 

TABLE  29. 

p.  ct. 

Distilled  water 1,280 

Potassium  nitrate 1 , 060 

Potassium  nitrate,  citric  acid,  0.01  M 802 

Citric  acid,  0.01  N 604 

Potassium  hydroxid,  0.01  M 604 

Effects  similar  to  the  original  are  to  be  discerned  in  the  above.  The 
combination  of  acid  and  salt  reduces  the  hydration  capacity  of  the 

1  Fenn,  W.  O.  Similarity  in  the  behavior  of  protoplasm  and  gelatine.  Proc.  Nat.  Acad.  Sci. , 
2:  539.  1916. 

1  MacDougal,  D.  T.,  and  H.  A.  Spoehr.  The  effects  of  acids  and  salts  on  biocolloids.  Science, 
46:269.  1917. 


44  Hydration  and  Growth. 

colloid  below  that  in  the  salt  alone.  A  wide  variety  of  tests  which  gave 
opportunity  for  comparisons  are  described  throughout  this  volume, 
but  a  few  may  be  recorded  here  which  were  carried  out  expressly  to 
obtain  evidence  on  this  point  with  biocolloids  of  different  constitution. 
Nucleinic  acid  is  a  constituent  of  the  nucleus,  and  as  the  only  other 
substance  from  this  body  which  had  been  introduced  into  the  tests 
which  could  be  assigned  to  the  nucleus  was  peptone,  its  swelling  re- 
actions were  tested  with  much  interest.  The  results  of  swellings  of 
these  substances,  when  combined  in  proportion  of  10  parts  nucleinic 
acid  to  90  parts  agar,  are  given  in  table  30. 

TABLE  30. 

p.  ct.  p.  ct. 

Distilled  water 1,400  1,025 

Potassium  nitrate,  0.01  M 900  800 

Potassium  nitrate,  citric  acid,  0.01  N 650  675 

Potassium  citrate,  0.01  N 850  750 

Potassium  citrate,  citric  acid,  0.01  N 725         

Citric  acid,  0.01  N 700  625 

Sodium  hydroxid,  0.01  M 1,000  925 

A  second  series  a  week  later  gave  measurements  shown  in  table  31. 

TABLE  31. 

p.  ct.  p.  ct. 

Distilled  water 950  1, 100 

Potassium  nitrate,  0.01  M 650  550 

Potassium  nitrate,  citric  acid,  0.01  N 575  500 

Citric  acid,  0.01  N 575  450 

Potassium  nitrate,  potassium  hydroxid,  0.01  N 900  750 

Potassium  hydroxid,  0.01  M 850  800 

This  mixture  is  seen  to  swell  most  in  distilled  water,  while  the  pro- 
portionate swelling  in  hydroxid  is  very  high,  being  greater  than  that  in 
the  salts  tested  or  in  acid.  Next,  it  is  apparent  that  the  two  potassium 
salts  produce  or  allow  an  amount  of  imbibition  not  very  much  short 
of  that  in  the  hydroxid.  The  acidification  of  the  salts  practically 
reduces  the  swelling  to  the  proportion  displayed  by  the  acid  alone. 
This  must  be  taken  to  apply  to  this  set  of  combinations  only.  It  may 
not  be  assumed  that  a  similar  generalization  would  hold  for  calcium. 

Following  the  above,  plates  composed  of  90  parts  agar  and  10  parts 
glycocoll,  0.15  mm.  in  thickness,  were  tested  in  series  parallel  to  the  above 
in  the  dark  chamber  at  16°  C.  The  swellings  are  given  in  table  32. 

TABLE  32. 

p.  ct.  p.  ct. 

Distilled  water 1,300  1,266 

Potassium  nitrate,  0.01  M 700  1,000 

Potassium  nitrate,  nitric  acid,  0.01  N 900  766 

Citric  acid,  0.01  N 666  766 

Sodium  hydroxid,  0.01  M 366  300 

The  effects  of  the  acidified  salt  and  of  the  acid  are  of  the  kind  pre- 
viously noted.  The  most  prominent  feature  of  the  reactions  was  the 
low  hydration  capacity  in  the  alkaline  solution  and  the  relatively  high 


Effect  of  Salts  and  Adds  on  Biocolloids  and  Cell-masses.  45 

expansion  in  acids,  the  action  of  the  solution  being  supplemented  by 
the  amino-acid  in  the  sections,  in  a  manner  similar  to  that  of  such  other 
amino-acids  as  aspartic  acid,  cystin,  tyrosin,  etc. 

A  mixture  of  agar  (50  parts)  and  gelatine  (50  parts)  poured  on 
Pratt-Dumas  brown  filter-paper  dried  to  a  total  thickness  of  0.3  mm. 
Swellings  of  this  were  made  in  the  dark  chamber  at  a  temperature  of 
16°  C.,  with  the  results  shown  in  table  33. 

TABLE  33. 

p.  ct. 

Distilled  water 400 

Potassium  nitrate 375 

Potassium  nitrate,  citric  acid,  0.01  M 350 

Citric  acid,  0.01  N 300 

Potassium  hydrate,  potassium  nitrate,  0.01  M 425 

Potassium  hydroxid,  0.01  M 325 

These  results,  so  far  as  they  may  be  correlated  with  the  earlier  ones, 
show  an  unexpected  relation  to  acid,  hydroxid,  and  water.  It  is  to  be 
noted  in  addition,  however,  that  a  combination  of  potassium  hydrate 
and  potassium  nitrate  gives  the  maximum  effect  in  the  series. 

The  application  of  parallel  tests  to  growing  tissues  is  complicated 
by  the  fact  that  varying  quantities  of  normal  salts  and  acid  salts  may 
be  present,  giving  a  buffer  effect.  The  concentration  of  the  hydrogen 
ion  may  be  determinable  by  estimation  of  the  titrable  acid  and  the 
dissociated  malates,  for  example,  as  found  by  Jenny  Hempel,  but  in 
addition  there  are  to  be  considered  the  effects  of  the  amino-acids  and 
amines,  which  are  not  easily  to  be  measured. 

Leaves  of  Mesembryanthemum  edule,  which  were  not  yet  fully  grown, 
were  cut  into  sections  about  a  centimeter  long  and  allowed  to  dry 
in  the  air.  When  the  greater  part  of  the  water  had  been  lost  and  the 
sections  had  a  leathery  consistency,  one  of  the  angles  was  removed 
with  the  scissors,  leaving  a  specimen  1.8  mm.  thick.  The  preparations 
were  by  no  means  uniform.  The  use  of  three  to  obtain  each  record 
would  tend  to  obviate  or  smooth  the  discrepancies,  but  the  data  given 
in  table  34  can  not  be  taken  as  having  been  obtained  from  preparations 
strictly  equivalent. 

TABLE  34. — Swelling  of  sections  of  Mesembryanthemum. 

p.  ct. 

Distilled  water 72 

Potassium  nitrate,  0.01  M 69 

Potassium  nitrate,  0.1  M 56 

Citric  acid,  potassium  nitrate,  0.01  M 44 

Citric  acid,  0.01  N 72 

Sodium  hydroxid,  0.01  M 28 

The  main  interest  in  this  set  of  reactions  is  that  directed  to  the 
comparison  of  the  swellings  in  potassium  nitrate,  citric  acid,  and  the 
combination  at  the  same  concentration.  The  coefficient  of  swelling 
in  the  plant-like  sections  of  agar-oat  protein  and  agar-bean  protein  was 
least  in  the  acidified  salt  solution,  although  the  hydrogen-ion  concen- 


46  Hydration  and  Growth. 

tration  of  the  combined  solution  should  be  equivalent  to  that  of  the 
salt  alone.  The  combination  of  citric  acid  and  potassium  nitrate  is 
open  to  some  objection,  but  the  effects  described  are  similar  to  those 
obtained  by  the  use  of  potassium  chloride  and  hydrochloric  acid. 

The  total  acidity  of  pure  juice  of  fresh  material  at  Tucson  varied 
from  0.0280  in  the  morning  to  0.0232  per  centimeter  0.01  N  of  sodium 
hydoxid  at  4h30m  p.  m.  Probably  some  of  the  acid  was  broken  up 
during  the  drying,  but  the  cell  colloids  would  still  be  decidedly  acid. 
The  hydrogen-ion  concentration  in  another  species  of  Mesembryan- 
themum  determined  by  Lakmoid  tests  and  electrometer  measurements 
by  Hempel  gave  values  of  pH  — 4.8  to  5.2  for  the  first  and  4.61  to  4.84 
by  the  second  method. 

The  matter  was  given  further  test  by  taking  sections  of  young  leaves 
in  a  flaccid  condition  and  measuring  the  total  swellings,  which  are 

given  in  table  35. 

TABLE  35. 

p.  ct. 

Water 22 

Potassium  nitrate, 0.01  M 22 

Potassium  nitrate,  citric  acid,  0.01  N 17 

Citric  acid,  0.01  N 17 

Sodium  hydroxid 11 

The  swelling  in  potassium  nitrate  agreed  with  that  of  the  dried 
material  in  being  nearly  equivalent  to  that  hi  distilled  water,  and  less 
in  acid  salt  solution,  but  the  swelling  in  acid  is  less,  the  proportionate 
swelling  in  hydroxid  being  about  the  same. 

The  effects  of  a  similar  series  of  reagents  were  tried  upon  disks  from 
growing  joints  of  Opuntia,  with  the  results  given  in  table  36. 

TABLE  36. 

p.  ct. 

Distilled  water 11 

Potassium  nitrate,  0.01  M 9 

Potassium  nitrate,  citric  acid,  0.01  N 10 

Citric  acid,  0.01  N 9 

Potassium  nitrate,  potassium  hydroxid,  0.01  M 10 

The  relations  here  are  different  in  character  from  those  exhibited 
by  the  material  previously  examined,  but  the  departures  are  so  small 
that  no  safe  conclusion  may  be  founded  on  them. 

The  incorporation  of  any  salt  with  a  colloid  in  the  disperse  phase 
would  of  course  allow  the  formation  of  adsorption  compounds  to  an 
extent  and  of  a  kind  not  possible  when  the  salt  enters  the  hydrating 
gel  in  a  solution.  The  hydration  of  such  a  salted  colloid  might  be 
expected  to  take  place  at  a  different  rate  and  to  a  total  varying  from 
that  of  the  unsalted  colloid  with  unsatisfied  chemical  affinities  and 
under  the  conditions  of  surface  tension  which  would  prevail.1 

1  Loeb,  J.     The  similarity  of  the  action  of  salts  upon  the  swelling  of  animal  membranes  and  of 
powdered  colloids.     Jour.  Biol.  Chem.,  31:  343.     1917. 


Effect  of  Salts  and  Adds  on  Biocolloids  and  Cell-masses.  47 

The  first  tests  of  the  effects  of  incorporated  salts  were  those  in 
which  the  conditions  of  the  plant  were  simulated,  and  the  most  rational 
procedure  seemed  to  be  one  in  which  the  culture  salts  of  plants  should 
be  added  to  a  mixture  of  agar  and  bean  protein,  the  proportions  being 
as  follows : 

TABLE  37. 

gm. 

Agar 9 

Bean  protein 1 

Potassium  nitrate 0.00506 

Di-potassic  phosphate .01622 

Magnesium  sulphate .  03660 

Calcium  nitrate .  03490 


Total 10.09278 

This  material  was  reduced  to  a  dried  plate  0.18  mm.  in  thickness, 
which  was  swelled  under  the  auxograph  at  a  temperature  of  15°  C., 
giving  increases  of  1,400  to  1,500  per  cent  in  distilled  water,  as  might 
be  contrasted  with  2,100  to  about  2,600  per  cent  in  the  reactions  of 
similar  sections  free  from  salts. 

A  second  preparation  was  made,  but  with  ten  times  the  amount  of 
salt  used  in  the  first  one,  the  salts  forming  nearly  9  per  cent  of  the 
dry  weight  in  one  case  and  0.85  per  cent  in  the  other.  The  swellings 
of  the  biocolloid  with  the  higher  salt-content  are  given  below: 

TABLE  38.  p.  ct. 

Distilled  water 958 

Citric  acid 361 

Potassium  hydroxid, 528 

Potassium  nitrate,  citric  acid,  0.01  N 389 

Potassium  nitrate,  0.01  M 694 

Potassium  hydroxid,  potassium  nitrate,  0.01  M 472 

The  large  proportion  of  salts  is  seen  to  hinder  swelling  in  a  notable 
manner.  Temperature  effects  are  of  great  importance  in  this  con- 
nection, as  it  was  found  that  sections  of  the  plates  which  contained  the 
lesser  proportion  of  culture  salts  and  which  increased  1,325  per  cent 
in  distilled  water  at  15°  C.,  swelled  2,666  per  cent  at  48°  to  40°  C.  (See 
Chapter  IX  for  a  fuller  discussion  of  temperature  effects.) 

Another  set  of  dried  plates  was  made  for  the  purpose  of  obtaining 
comparisons  in  two  directions.  The  colloidal  constituents  of  the 
mixture  were  extended  to  include  agar  70,  dextrose  5,  gelatine  5,  pep- 
tone 5,  asparagine  5,  nucleinic  acid  5,  and  bean  protein  5  parts,  and  a 
set  of  dried  plates  was  made  up  as  above  in  distilled  water.  A  second 
set  was  made  up  in  the  culture  solution,  in  which  the  salts  amounted 
to  0.85  per  cent  of  the  dry  weight.  The  swellings  were  made  on  two 
successive  days  in  a  chamber  constant  at  15°  C.  It  is  to  be  noted  that 
in  any  inspection  of  these  results  rigid  comparisons  may  not  be  allowed 
between  the  swellings  of  the  two  kinds  of  plates  in  any  solution.  The 
basis  of  all  comparisons  must  be  the  ratio  of  the  swelling  of  each  plate 
in  any  solution  to  its  swelling  in  distilled  water.  (See  table  39). 


Hydration  and  Growth. 
TABLE  39. 


Salt-free. 

Salted. 

Distilled  water  

p.  ct. 
1,250 

p.  ct. 
500 

Potassium  nitroxid,  0.01  M  

875 

550 

Potassium  nitroxid,  citric  acid,  0.01  N  

535 

425 

Citric  acid  0.01  N  

446 

425 

Potassium  hydroxid,  potassium  nitrate,  0.01  M 
Potassium  hydroxid,  0.01  M  

750 
535 

550 
525 

The  relative  swellings  of  the  biocolloid  without  the  culture  salts 
presents  the  general  features  of  such  mixtures,  being  highest  in  dis- 
tilled water,  next  in  potassium  nitrate,  less  hi  acidified  potassium 
nitrate,  less  in  citric  acid,  and  varying  in  the  relations  of  the  hydroxid 
and  the  alkaline  salts. 

The  plates  in  which  the  culture  salts  were  incorporated  showed 
relative  swellings  which  did  not  differ  widely  from  the  expectancy, 
except  in  the  swelling  in  alkali  and  alkaline  salts.  The  addition  of  the 
dextrose  could  not  be  seen  to  exert  any  definite  action.  The  outstand- 
ing fact  is  the  general  retarding  effect  of  salinity  on  the  hydration 
capacity,  a  fact  of  possible  enormous  importance  in  the  organism. 

A  mixture  including  4  parts  of  agar,  5  parts  of  gum  arabic,  and  1 
part  of  gelatine  was  made  and  sufficient  potassium  chloride  was  added 
to  make  it  0.01  M  of  this  compound.  Swellings  at  18°  to  20°  C.  were 

as  given  in  table  40. 

TABLE  40.  p.  ct. 

Distilled  water 612 

Citric  acid,  0.01  N 465 

Sodium  hydroxid,  0.01  M 214 

Hydrochloric  acid  0.01  M 535 

Hydrochloric  acid,  potassium  chloride,  0.01  M 419 

A  general  restriction  of  nearly  all  of  the  swelling  reactions  is  illus- 
trated by  the  measurements  in  table  40,  while  the  relative  increase  in 
acids  is  high. 

As  a  further  combination  of  two  forms  of  carbohydrate,  albumen, 
amino-acids,  and  of  the  salts  which  are  found  in  plants,  a  mixture  was 
made  which  contained  the  following  material : 

TABLE  41.  gm. 

Agar 6 

Acacia 2 

Gelatine 1 

Albumin  (Phaseolua) 1 

Potassium  nitrate 0 . 0058 

Potassic  phosphate,  dibasic 0185 

Magnesium  sulphate  (7H2O) 0418 

Calcium  nitrate  (4H2O) 0398 


Total  colloid  material 10 

Total  nutrient  salts ...  .  0105 


Effect  of  Salts  and  Adds  on  Biocolloids  and  Cell-masses. 


49 


I2p.m.      m.       12p.m.    m.       Igp^m. 


I2p.n 


I2p.m.     m.       12p.m. 


Dried  plates  were  made  up  in  the  usual  manner  and  freed  from 
water  in  a  special  chamber  with  fan  at  a  temperature  of  about  16°  C. 
Swellings  at  14°  to  17°  C.  were  as  shown  in  table  42. 

TABLE  42.  p.  ct.  p.  ct. 

Distilled  water 2,200  2,240 

Citric  acid  0.01  N 802  880 

Sodium  hydrate,  0. 01  M 602  680 

Potassium  chloride,  hydrochloric  acid,  0.01  M 700  640 

The  notable  feature  is  the  high  swelling  in  water  and  the  fact  that 
the  increase  in  the  acid  solution  is  less  than  in  the  alkaline,  facts 
which  are  probably  due  in  part  to  the  action  of  gum  acacia.  This 
set  of  swellings  has  unusual  interest  because  of  its  composition,  in 
which  the  categories  of  substances  in  the  plant  are  represented  with 
some  fairness  and  adequacy  (fig.  9).  FIG.  9. 

Tracing  of  auxographic 
records  of  swelling  of 
sections  of  plates 
consisting  of  6  parts 
agar,  2  parts  gum  ar- 
abic,  1  part  gelatine, 
1  of  bean  albumin 
and  .05  nutrient  salts 
in  water.  A,  citric 
acid. 01  N.  .B, sodium 
hydrate.  01  M.  C,  po- 
tassium chloride  and 
hydrochloric  acid 
.01  M.  D,  at  14°  to 
17°  C.  Downward 
course  of  pen  tracing 
denotes  increase  as 
indicated  by  numer- 
als on  margin. 

The  results*obtainedTin*some'*of  the  foregoing  experiments  indicated 
that  the  treatment  of  the  biocolloid  with  salts  before  acid  solutions 
were  applied  might  show  some  features  of  importance,  and  this  was 
also  supported  by  the  alternating  effects  described  in  detail  elsewhere 
in  this  volume.  Plates  had  been  made  of  agar  90  parts  and  bean 
protein  10  parts  in  two  sets.  In  one  a  culture  solution  was  used  in 
such  concentration  that  the  included  salts  formed  0.85  per  cent  of  the 
dry  weight  of  the  sections.  In  the  other  the  concentration  of  the 
salts  was  about  ten  times  this  amount.  Both  showed  opaque  dots  or 
minute  regions,  supposedly  insoluble  globulin. 

Swellings  were  made  at  temperatures  of  16°  to  17°  C.  on  October  12 
and  13,  1917,  and  the  measurements  obtained  from  the  sections  con- 
taining the  larger  proportion  of  salts  are  as  given  in  table  43. 

TABLE  43. 

p.  ct. 

Distilled  water 522 

Citric  acid,  0.05  N 583 

Citric  acid,  0.05  N 413 

Citric  acid,  0.0005  N 348 

Citric  acid,  0.00005N 500 


\\ 


50  Hydration  and  Growth. 

The  measurements  in  table  43  were  taken  at  the  end  of  30  hours, 
at  which  time  expansion  was  not  complete,  although  further  swelling 
would  not  materially  alter  the  relative  values.  The  proportion  of 
salts  actually  present  was  about  one-twelfth  of  the  biocolloid,  which  in 
volume  amounted  to  about  12  c.  mm.  The  dishes  held  30  c.  c.,  and 
from  these  data  it  may  be  possible  to  calculate  proportions  of  salts 
and  acids  for  comparison  with  the  cases  in  which  the  salts  are  applied 
in  solution. 

A  second  test  was  made  with  the  same  biocolloid  as  above,  but  to 
which  had  been  added  but  one-tenth  of  the  foregoing  proportion  of 
culture  salts.  The  plates  were  thinner,  but  the  swellings  were  made 
at  the  same  temperatures  and  under  approximately  the  same  con- 
ditions, with  results  as  follows: 

TABLE  44. 

p.  ct. 

Distilled  water 1,667 

Citric  acid,  0.05  N 667 

Citric  acid,  0.005  N 899 

Citric  acid,  0.0005  N 1 , 139 

Citric  acid,  0.00005  N 1,500 

The  swelling  of  these  plates,  which  were  0.18  mm.  in  thickness,  was 
carried  out  at  a  temperature  of  16°  to  17°  C.,  and  the  expansion  in 
distilled  water  shows  the  retarding  effect  of  the  salt  alone  when  com- 
parison is  made  with  agar-bean  protein  mixtures  not  treated  with  the 
nutrient  solution.  The  citric  acid  in  its  heaviest  concentration  is 
equivalent  to  the  strongest  solution  encountered  in  plants  in  this  work, 
at  which  it  is  seen  to  retard  swelling  very  much.  Reduction  of  the  con- 
centration of  the  salts  seems  to  be  followed  by  a  proportionate  increase 
of  water-capacity,  in  a  fairly  regular  manner.  The  discrepancy  in  the 
swelling  in  0.005  N  acid  in  the  more  heavily  salted  plates  is  probably 
an  instrumental  error  and  will  be  so  considered  until  confirmed.  In 
the  most  attenuated  solution  of  acid  the  swelling  approaches  that  of 
distilled  water,  in  which  the  salt  effect  alone  is  apparent. 

The  series  of  increases  of  a  biocolloid  given  on  page  48  present  the 
general  differences  and  relations  of  sections  from  plants,  and  these, 
rather  than  the  one  in  table  44,  which  has  such  a  high  swelling  coeffi- 
cient in  water,  are  of  the  character  more  usually  encountered  in  cell- 
masses.  It  is  to  be  recalled,  however,  that  the  composition  of  the 
biocolloid,  including  a  mucilage,  albumin,  and  amino-acids,  is  one 
which  may  well  be  duplicated  in  the  plant,  and  it  may  be  the  recurrence 
of  such  combinations  which  would  furnish  the  phenomena  so  prominent 
in  the  involutions  of  the  cell. 

The  principal  deductions  of  the  present  work  support  the  conclusion 
that  agencies  or  conditions  which  increase  the  hydration  capacity  of 
protoplasm  accelerate  growth,  and  any  factor  which  tends  to  lessen 
either  the  rate  of  absorption  or  the  total  hydration  capacity  of  living 


Effect  of  Salts  and  Adds  on  Biocolloids  and  Cell-masses.  51 

matter  retards  and  limits  growth  and  development,  or  may  have  such 
special  effects  as,  for  example,  the  condensation  of  chromatin  into 
special  masses  or  chromosomes  in  the  course  of  cell  division.1  Now, 
protoplasm  parallels  the  colloidal  action  of  gelatine  only  in  so  far  as  it 
is  composed  of  protein  or  protein  derivatives,  and  the  proportions  of 
these  substances  and  of  the  associated  carbohydrates  vary  from  organ 
to  organ  and  with  the  course  of  the  seasons  or  the  stage  of  develop- 
ment. Furthermore,  the  biocolloids  of  the  cell  are  acidified  or  salted, 
and  their  behavior  toward  any  reagent  externally  applied  will  of 
course  be  determined  or  modified  by  all  of  the  chemical  and  adsorptive 
relations  implied.  Lastly,  the  residual  acids  of  respiration  vary  from 
hour  to  hour  under  ordinary  circumstances. 

It  might  be  expected,  therefore,  that  tests  which  are  planned  to 
determine  the  influence  of  acids  or  bases  on  growth  would  bring  out  a 
diversity  of  results. 

G.  A.  Borowikow  (Borovikov),  a  Russian  working  at  the  University 
of  Odessa,  used  seedlings  of  Helianthus  6  days  old  for  testing  the 
effects  of  acids  and  salts  upon  growth.2  The  roots  of  the  seedlings 
were  immersed  in  water  and  solutions  in  glass  jars  and  the  effects 
upon  growth  derived  from  measurements  of  the  length  of  the  plant- 
lets.  Acceleration  and  final  maxima  were  obtained  by  the  use  of 
hydrochloric,  sulphuric,  nitric,  acetic,  and  boric  acids,  in  the  order 
named,  that  of  hydrochloric  being  the  greatest  as  compared  with  the 
growth  of  plants  in  distilled  water.  It  was  also  noted  that  the  addi- 
tion of  salts  to  the  acids  influenced  the  rate  and  final  effect,  according 
to  the  character  of  the  base.  Salts  with  weak,  easily  hydrolyzable 
bases  affected  growth  almost  solely  according  to  the  concentration 
of  the  hydrogen  ions,  but  the  stronger  bases  exercised  a  definite 
effect,  which  in  this  author's  work  was  to  retard  growth.  It  is  clear 
that  the  growing  cell-masses  of  Helianthus  are  not  identical  in  their 
action  with  such  amphoteric  colloids  as  gelatine. 

Sections  of  growing  internodes  of  Helianthus  in  my  own  work  did 
not  show  their  greatest  swelling  in  simple  acid  solutions,  but  in  those 
to  which  some  salt  of  the  same  molecular  concentration  had  been 
added.  It  was  also  found  that  the  hydration  capacity  of  artificially 
mixed  biocolloids,  dried  and  living  sections  of  plants  showed  a  decrease 
in  hydration  capacity  with  rise  in  temperature  above  16°  to  18°  C.  in 
acid  solutions.  On  the  other  hand,  it  has  been  shown  that  in  the 
petiole  of  the  calla  (Richardia} ,  which  attains  a  length  of  over  half 
a  meter  and  continues  to  elongate  at  varying  rates  throughout  its 
entire  length,  the  greatest  acidity  is  in  the  region  of  most  rapid  growth. 
It  is  evident  that  interpretations  must  take  into  account  a  wider 

1  Mathews,  A.  P.     Physiol.  Chem.,  2d  ed.,  p.  235.     1916. 

2  Borowikow,  G.  A.     Ueber  die  Ursachen  dea  Wachstums  der  Pflanzen.     Biochem.  Zeitschr., 
48:  230,  and  50:  119.     1913. 


52  Hydration  and  Growth. 

range  of  conditions  than  those  presented  by  the  swelling  of  simple 
gelatine  in  electrolytes,  especially  at  uncontrolled  or  unrecorded  tem- 
peratures. 

Professor  F.  E.  Lloyd,  working  under  the  equable  temperature  con- 
ditions of  the  Coastal  Laboratory  at  Carmel,  California,  measured 
the  growth  of  pollen-tubes  of  Phaseolus  odoratus  in  acids  (hydro- 
chloric, acetic,  malic,  citric,  formic,  and  oxalic)  at  concentrations 
N/200  to  N/25,600  in  association  with  cane  sugar  in  concentration 
of  40  per  cent.  In  these  solutions  no  growth  occurs  at  concentra- 
tions at  or  above  N/3,200  of  the  acid  component.  Below  that  limit 
the  rate  of  growth  is  inversely  as  the  concentration.  The  rate  and 
total  amount  of  growth  possible  for  any  concentration  varied  with 
the  acid,  it  being  least  at  the  higher  concentrations  for  formic  and 
oxalic  and  highest  for  acetic.1 

No  temperature  records  are  cited  in  any  of  these  cases  or  in  the  other 
work  described  by  Long  or  Dachnowski. 

The  measurements  of  E.  R.  Long  brought  out  the  fact  that  growth 
in  Opuntia  was  greater  in  culture  salt  solutions  than  in  any  other 
medium  tested,  while  that  in  alkaline  solution  was  more  than  in  either 
malic  or  hydrochloric  acid,  these  substances  being  used  in  concentra- 
tions of  N/50,  while  the  solutions  of  Borowikow  were  N/100  or  weaker.2 

A.  Dachnowski  measured  the  swelling  and  water-relations  of  seeds 
of  beans  and  corn  and  cuttings  of  tomato  shoots  and  obtained  some 
facts  of  great  interest.3  Beans  were  found  to  absorb  and  retain  less 
water  in  acid  solutions  (N/800)  than  in  equi-nonnal  alkaline  solutions. 
The  cations  Ca  and  Na  were  more  active  than  potassium  in  limiting 
imbibition  hi  beans,  a  relation  which  is  reversed  hi  corn  grams,  in 
which  the  greatest  swelling  took  place  in  calcium,  a  lesser  amount  in 
potassium,  and  a  minimum  in  sodium.  Cuttings  of  tomato  plants 
were  found  to  function  better  hi  an  alkaline  medium  than  an  acid,  and 
best  of  all  in  water  in  absorption  and  transpiration.  Sulphuric  acid 
at  N/3,200  and  potassium  hydroxid  at  N/6,400  furnished  exceptions  to 
these  conclusions.  The  effect  of  any  salt  on  the  water-relations  of  the 
plants  used  was  the  sum  of  the  constituent  ions,  a  conclusion  confirm- 
ing the  work  of  Borowikow. 

The  well-known  cultural  conditions  required  by  many  bacteria  and 
fungi  furnish  still  further  exemplification  of  the  diversity  of  behavior 
of  the  biocolloids  in  the  hydration  necessary  for  growth.  The  plasma 
of  bacteria  is  high  in  albumin  and  many  bacteria  show  the  highest 
velocity  and  greatest  development  in  a  medium  containing  the  soluble 
proteins  combined  with  sodium  chloride  and  brought  to  an  alkaline 

1  Lloyd,  F.  E.  Collodial  phenomena  in  the  protoplasm  of  pollen  tubes.  Kept.  Dept.  Bot- 
Res.,  Year  Book  Carnegie  Inst.  Wash.,  1917,  p.  63. 

*Long,  E.  R.     Growth  and  colloid  hydratation  in  cacti.     Bot.  Gazette,  59:  491.     1915. 

1  Dachnowski,  A.  The  effects  of  acid  and  alkaline  solutions  upon  the  water-relations  and  the 
metabolism  of  plants.  Amer.  Jour.  Bot.,  1 :  412-439.  1914. 


Effect  of  Salts  and  Adds  on  Biocolloids  and  Cell-masses.  53 

condition  with  sodium  bicarbonate.  The  sodium  albuminates  which 
would  be  formed  in  the  medium  are  highly  dissociated,  as  would  be  the 
biocolloids  of  the  cell,  and  both  diffusion  and  hydration  would  be 
accelerated.  This  supposition  would  be  based  on  the  inference  that 
the  amphoteric  cell-proteins  were  stronger  acids  than  bases.  A  further 
result  of  the  conditions  of  bacterial  action  is  to  be  seen  in  the  fact  that 
the  growth  of  bacteria  in  cultures  containing  carbohydrates  is  accom- 
panied by  the  production  of  an  acidity  which  checks  then1  growth  and 
retards  their  action  upon  proteinaceous  substances  present.  On  the 
other  hand,  many  molds  grow  best  in  relatively  high  concentrations 
of  acids,  the  cycle  of  fermentations  in  milk  furnishing  a  striking  example 
of  the  differential  action  of  these  organisms.1  The  first  stage  is  charac- 
terized by  the  action  of  the  lactic-acid-producing  bacteria,  which  con- 
tinue until  their  products  reach  an  inhibitory  concentration.  The 
soured  milk  now  becomes  a  suitable  medium  for  Oidium  and  Penicil- 
lium,  which  may  thrive  in  a  solution  containing  as  much  as  1.25  per 
cent  lactic  acid,  and  continue  to  grow  until  the  acid  is  exhausted,  and 
then  the  neutral  or  alkaline  solution  again  becomes  a  suitable  habitat 
or  bacteria. 

German,  N.,  and  L.  F.  Rettger.    The  influence  of  carbohydrate  on  the  nitrogen  metabolism  of 
bacteria.     Jour,  of  Bacter.,  3:  389.     1918. 


V.  THE  EFFECTS  OF  ORGANIC  ACIDS  AND  THEIR  AMINO 
COMPOUNDS  ON  HYDRATION  AND  GROWTH. 

The  biocolloids  of  the  plant  are  pentosan-protein  mixtures  in  which 
the  substances  of  these  two  main  groups  vary  widely  in  their  propor- 
tions, with  a  smaller  proportion  of  lipins  probably  more  or  less  localized. 
The  variables  are  so  large  that  generalizations  concerning  the  action 
of  the  plasmatic  mass  are  not  easily  to  be  founded.  Of  the  more  im- 
portant assertions  concerning  the  action  of  protoplasm,  the  earliest 
and  most  widely  used,  that  protoplasm  undergoes  hydration  like  an 
amphoteric  colloid,  and  is  exemplified  by  swelling  gelatine,  has  long 
since  failed  to  satisfy  the  experimental  conditions  or  to  offer  parallels 
to  the  action  of  cell-masses  of  the  higher  plants. 

It  obviously  follows  that  the  assumption  adopted  by  many  writers 
that  any  conditions  which  facilitate  the  ionization  of  the  proteins 
accelerate  growth  is  not  tenable,  since  the  effect  of  acidity  is  to  lessen 
hydration  of  the  pentosans  or  pentosan  mixtures  or  cell-masses  when 
acting  directly  or  in  the  presence  of  salts.  The  extensive  use  of  agar 
to  represent  the  pentosan  element  in  biocolloids  in  my  experiments 
does  not  imply  that  this  substance  or  any  other  body  presenting  all 
of  its  main  physical  characters  are  invariably  present  in  the  cell.  The 
gums  from  acacia,  tragacanth,  Opuntia,  and  from  Prosopis  and  the 
cherry-tree  in  all  probability  represent  types  of  pentosans  which  may 
really  be  the  most  abundant  in  plants.  These  gums  or  mucilages  are 
readily  dispersible  and  have  an  indefinite  hydration  capacity  which 
soon  passes  beyond  the  limits  of  measurement  by  the  auxograph 
Their  water-absorbing  capacity  would  be  none  the  less  important, 
when  inclosed  in  the  cell-sacs.  The  solubility  of  protoplasm  has 
formed  the  subject  of  some  discussion  among  cytologists,  and  it  would 
seem  highly  probable  that  valid  observations  of  both  extremes  may 
have  been  made,  the  matter  depending  chiefly  on  the  nature  of  the 
pentosan,  gum,  or  mucilage  which  entered  into  the  plasmatic  colloids, 
together  with  the  character  of  the  more  liquid  phase,  or  the  cell-sap. 

The  conclusions  of  Loeb1  to  the  effect  that  it  is  not  possible  for  an 
amphoteric  colloid  to  be  acted  upon  by  both  ions  at  the  same  time, 
if  correct,  would  add  still  further  proof  to  the  fact  that  protoplasm 
does  not  behave  like  an  amphoteric  colloid  in  its  hydration  relations, 
except  in  so  far  as  it  may  be  predominantly  composed  of  such  material. 

Data  for  a  critical  review  of  this  matter  are  not  available  at  present, 
but  it  is  known  that  bacteria  are  high  in  albumin,  and  similar  richness 
of  proteins  is  exhibited  by  fungi,  and  that  certain  algae  show  a  large 
proportion  of  amino-acids.  In  addition,  proteins  are  abundant  in 

1Loeb,   J.     Amphoteric   colloids.     Chemical   influence   of    the   hydrogen-ion    concentration. 
Jour.  Gen.  Physiol.,  1:  39.     1918. 

54 


Effect  of  Certain  Organic  Acids  and  Amino  Compounds.  55 

reproductive  elements,  while  voluminous  notices  of  these  substance 
and  their  derivatives  are  found  in  cytological  literature,  many  of  which, 
however,  need  verification  and  analysis  to  make  them  of  value. 

If  protoplasm  were  entirely  or  dominantly  proteinaceous,  the  actual 
acidity  or  hydrogen-ion  concentration  of  the  sap  might  be  taken  as  the 
chief  factor  in  maintaining  the  rate  and  determining  the  course  of 
hydration  and  growth.  The  predominance  of  the  pentosans  in  plant 
cells,  however,  offers  a  set  of  conditions  much  more  complex  than  that 
of  the  comparatively  simple  ionization  of  gelatine,  for,  as  has  been 
noted,  the  conditions  which  facilitate  the  action  of  protein  gels  retard 
and  limit  the  hydration  of  the  carbohydrate  gels  to  an  extent  and  in  a 
manner  which  depend  upon  the  structure  and  character  of  the  pen- 
tosans present. 

It  has  already  been  shown  that  pentosan  colloids  show  maximum 
hydration  capacity  in  the  presence  and  under  the  action  of  certain 
amino-compounds,  a  subject  to  which  the  larger  part  of  this  chapter 
will  be  given. 

The  actual  acidity,  or  hydrogen-ion  concentration  of  the  sap,  is 
widely  different  from  the  total  amount  of  acid,  as  some  is  always  com- 
bined with  such  bases  as  potassium,  sodium,  calcium,  magnesium, 
iron,  and  aluminium,  making  a  "buffer"  by  which  the  degree  of  dis- 
sociation is  controlled  within  certain  limits.  This  range  of  variation 
as  it  appears  in  separate  estimations  is  rather  large  as  compared  with 
variations  in  animals.  It  is  necessary  to  bear  in  mind,  however,  that 
a  cell-mass  is  not  uniformly  acid  or  that  the  entire  mass  of  the  cell- 
colloids  is  saturated  with  a  solution  of  the  same  concentration. 

In  any  buffer  situation,  however,  a  lessening  of  the  hydrogen-ion 
concentration  of  the  sap  would  be  followed  by  increased  dissociation 
of  the  acid  radicle  of  the  salts,  and  increase  of  acidity  beyond  a  certain 
point  would  result  in  a  reversal  of  the  process.  The  actual  acidity  is 
expressed  by  the  negative  common  logarithm  of  the  number  of  dis- 
sociated hydrogen  ions  given  as  the  value  of  Sorensen's  symbol  pH. 
This  may  vary  from  3.9  to  5.7  in  various  succulents  examined  by 
Jenny  Hempel,  and  may  approach  neutrality  at  pH-7  in  some  cases. 
A  singular  instance  of  wide  difference  between  actual  and  total  acidity 
is  offered  by  lemon  fruits,  the  sap  of  which  has  an  actual  acidity  of 
pH  =  2.3,  which  is  about  one-tenth  the  total  acidity,  which  may  be 
expressed  as  about  0.05  to  0.06  N.1 

The  variations  in  the  hydrogen-ion  concentration  of  the  cell-sap 
and  the  determination  of  the  agencies  which  may  cause  such  changes 
offer  a  most  inviting  field  for  research.2  In  a  recent  paper,  R.  B. 
Harvey  has  described  some  extremely  interesting  changes  in  the 
as  determined  by  potentiometer  methods,  of  cabbage  leaves  in  acidity, 

1  Hempel,  Jenny.     Buffer  processes  in  the  metabolism  of  succulent  plants.     Compt.  Rend, 
d.  trav.  d.  Lab.  Carlsberg,  13:  No.  1.     1917. 

2  Haas,  A.  R.     The  reaction  of  plant  protoplasm.     Bot.  Gazette,  63 :  232.     1917. 


56  Hydration  and  Growth. 

freezing,  and  finds  that  the  principal  effect  is  an  increase  in  the  hydro- 
gen-ion concentration  followed  by  a  general  return  to  original  values 
on  thawing,  with  changes  in  the  proteins  generally  consisting  in  pre- 
cipitations of  some  of  the  proteins.1 

The  results  of  Borowikow  and  those  of  Dachnowski  show  that  the 
growth  of  the  higher  green  plants,  does  not  depend  upon  the  hydrogen- 
ion  concentration  alone.  Acids  and  bases  both  influence  hydration 
and  growth.  In  addition  the  accelerating  effects  of  ammo-acids  and 
amines  on  hydration  of  biocolloids  and  cell-masses,  living  and  dead, 
go  far  to  support  the  conclusion  that  these  substances  facilitate  or 
increase  total  growth.  These  substances  are  built  up  from  simpler 
substances  in  the  plant  in  a  manner  which  is  by  no  means  clear,  al- 
though under  investigation  and  discussion  for  a  quarter  of  a  century. 
The  evidence  favors  the  assumption  that  they  come  together  in  the 
field  of  photosynthetic  activity.  The  structure  of  these  amino- 
groups  may  be  no  means  be  assumed  to  be  identical  with  that  of  the 
amino-acids  of  animal  metabolism,  in  which  they  occur  only  as  dis- 
integration products  of  the  proteins  or  albumins. 

The  total  amount  of  ammo-compounds  in  a  cell-mass  of  a  plant 
varies  widely  during  the  course  of  a  day,  and,  as  has  been  noted  above, 
the  proportion  of  nitrogenous  material  in  the  organs  of  the  cell  or  the 
members  of  a  shoot  may  be  greatly  different. 

As  the  hydrogen-ion  concentration  of  the  sap  is  known  to  remain 
fairly  constant,  as  the  salts  or  bases  which  affect  growth  also  change 
but  slowly,  attention  naturally  focuses  on  the  amino-compounds 
as  a  cause  in  modifying  the  rate,  course,  and  total  amount  of  growth. 
As  the  acids  and  their  salts  may  be  assumed  to  act  invariably  in  the 
presence  of  amino-groups,  a  series  of  tests  were  planned  which  should 
make  possible  a  comparison  of  the  action  of  some  of  the  commoner 
organic  acids  and  then*  amino-compounds. 

Two  groups  were  chosen  for  the  tests — succinic  acid  and  its  amino- 
compound,  amino-succinic  or  aspartic  acid,  which  are  dibasic;  and  its 
amide,  as  noted  above,  which  is  monobasic;  and  acetic  acid  and  its 
amino-compound,  glycocoll,  which  are  monobasic.  Sections  of  plates 
of  agar,  gelatine,  agar-gelatine,  agar-protein,  and  other  mixtures  were 
used.  Swellings  were  carried  out  in  the  equable  chambers  of  the 
Coastal  Laboratory,  at  15°  to  16°  C.  The  principal  results  are  given 
in  table  45. 

The  two  organic  acids,  succinic  and  acetic,  are  seen  to  exert  the 
classical  effect  on  gelatine,  the  greatest  hydration  taking  place  in  the 
higher  concentrations,  the  effect  decreasing  with  dilution  until  at 
0.0004  N  the  swelling  in  acetic  acid  was  scarcely  greater  than  in  dis- 
tilled water.  At  0.0004  M,  however,  the  dibasic  succinic  acid  showed 

1  Harvey,  R.  B.  Hardening  processes  in  plants  and  developments  from  frost  injury.  Jour. 
Agric.  Res.,  15:83.  1918. 


Effect  of  Certain  Organic  Acids  and  Amino  Compounds.  57 

a  swelling  less  than  that  in  distilled  water,  a  result  that  suggests  a 
rapid  solution  or  dispersion  from  the  surfaces  of  the  sections  and  alter- 
ations of  viscosity  in  the  mass. 

TABLE  45. — Hydration  of  agar,  gelatine,  agar-gelatine,  and  agar-oat  protein  in  organic  acids 
and  their  amino-compounds  at  16°  to  17°  C.     Expansion  in  percentages  of  dried  thickness. 


Concen- 
tration. 
Mol. 

Succinic 
acid. 

Aspartic 
acid. 

Aspar- 
agine. 

Acetic 
acid. 

Glyco- 
coll. 

p.  ct. 

p.  ct. 

p.  ct. 

p.  ct. 

p.  ct. 

0.3 

1,950 

.5 

1  060 

2  804 

.1 

1,000 

2,260 

1,333 

AOAB. 

.06 

1,091 

827 

2,308 

1,433 

.01 

1,273 

1,270 

2,365 

1,560 

2,965 

.002 

1,600 

1,400 

2,440 

1,790 

3,166 

.0004 

1,750 

1,788 

2,720 

1,955 

2,605 

.00008 

2,528 

2,080 

3,250 

2,640 

Water,  aver.  2,600  per  cent. 

0.1 

.05 

1,200 

1,500 

320 

952 

370 

GELATINE. 

.01 

700 

1,033 

480 

714 

.002 

500 

380 

500 

690 

360 

[ 

.0004 

433 

340 

467 

643 

360 

Water,  aver.  600  per  cent. 

0.5 

.1 

850 

AGAR  8  PARTS,  GELATINE 

.05 
.01 

716 
850 

910 
1,017 

1,485 
1,574 

850 
900 

1,233 
1,960 

2  PARTS. 

.002 

917 

1,295 

1,608 

922 

1,767 

.0004 

1,000 

1,667 

1,383 

1,117 

1,420 

.00008 

1,030 

1,786 

1,383 

1,167 

1,484 

Water,  aver.  1,684  per  cent. 

0  5 

500 

1 

809 

AGAR  8  PARTS,  OAT-PROTEIN 

.05 
.01 

700 
864 

855 
900 

1,867 
2,455 

1,090 
1,255 

1,983 
2,340 

PARTS. 

.002 

909 

1,670 

2,523 

1,738 

3,050 

.0004 

1,136 

2,600 

2,675 

2,238 

3,000 

.00008 

2,330 

3,050 

2,600 

2,480 

Water,  aver.  2,365  per  cent. 

Mixtures  of  agar  (8  parts)  and  gelatine  (2  parts)  were  now  tested, 
and  the  hydration  in  succinic  acid  at  0.00008  M  was  but  1,030  per 
cent,  as  compared  with  1,684  per  cent  in  water,  while  acetic  acid  was 
slightly  higher,  1,167  per  cent.  A  similar  statement  would  hold  for 
the  action  of  these  acids  on  agar  and  for  agar-protein,  the  hydration 
in  water  alone  being  reached  more  nearly*  than  in  the  agar-gelatine 
sections. 

When  we  now  turn  to  amino-succinic  or  aspartic  acid  and  amino- 
acetic  acid  or  glycocoll,  some  new  relations  are  uncovered.  The 
aspartic  acid  appeared  to  exercise  a  notable  influence  on  the  hydration 
of  agar.  The  limit  of  its  solubility  appeared  to  be  about  0.05  M 
at  15°  to  20°  C.  When  more  than  this  was  added  to  the  water 


58  Hydration  and  Growth. 

used  for  solution  a  swelling  in  excess  of  the  expectancy  resulted.  It 
was  also  seen  that  the  surface  of  the  liquid  became  covered  with  thin 
crystals.  In  all  probability  the  solution  or  dispersion  of  some  agar 
into  the  water  resulted  in  the  displacement  of  some  of  the  acid,  with 
the  result  that  the  sections  were  actually  hydrated  from  a  solution  less 
concentrated,  giving  a  swelling  in  excess  of  the  expectancy. 

Tests  were  now  made  at  the  same  temperature  and  under  the  same 
conditions  with  plates  consisting  of  agar  (8  parts)  and  gelatine  (2 
parts),  in  order  to  ascertain  the  results  when  the  carbohydrate  was  in 
colloidal  combination  with  complex  amino-compounds.  The  trios  of 
sections  had  shown  swellings  of  about  1,700  per  cent  in  distilled  water 
under  the  same  conditions  and  had  an  average  thickness  of  0.28  mm. 

TABLE  46. 

p.  ct. 

Aspartic  acid,  0.05  M 910 

Aspartic  acid,  0.0002  M 1 , 090 

Aspartic  acid,  0.00008  M 1,786 

The  effect  of  the  acid  is  seen  to  vanish  at  a  much  greater  concen- 
tration than  on  the  agar  alone,  the  swelling  at  saturation  being  about 
half  that  of  distilled  water. 

After  the  experience  noted  above,  new  plates  of  agar  (9  parts)  and 
aspartic  acid  (1  part)  were  made.  The  amino-acid  was  placed  in  the 
water  in  which  the  agar  was  liquefied  to  a  2.5  per  cent  solution.  The 
usual  translucency  of  the  agar  was  modified  to  a  pale  milky  appear- 
ance, and  its  viscosity  seemed  to  be  decreased.  Soon  after  setting, 
cracks  and  fractures  appeared  in  the  plates.  This  of  course  allowed 
shrinkage  in  the  long  axes  of  the  plates  and  would  make  it  impossible 
for  the  sections  to  swell  in  thickness  to  the  same  proportion  as  the 
coherent  agar  plates.  These  new  plates  came  down  to  a  thickness  of 
about  0.16  mm.  and  showed  swellings  of  220  per  cent  in  distilled  water 
at  16°  C.  and  of  a  slightly  greater  expansion  in  a  solution  of  0.05  M 
asparagin,  in  which  the  swelling  was  281  per  cent.  It  is  to  be  noted 
that  while  the  aspartic  acid  is  present  in  a  more  concentrated  condition 
in  these  plates  than  is  possible  in  water,  yet  the  entire  amount  was 
held  in  the  colloidal  mesh  or  plate  and  showed  no  formation  of  crystals 
on  the  surface  or  in  the  sections,  as  in  the  case  of  the  less-soluble  tyrosin. 
The  hydration  of  the  colloid  with  the  acid  incorporated  in  it  is  less 
than  that  which  may  take  place  when  the  acid  is  dissolved  to  saturation 
in  the  water  in  which  the  swellings  are  made.  The  influence  of  this 
acid  on  agar  was  not  widely  different  from  that  of  succinic  acid,  but  it 
caused  greater  swelling  in  equimolecular  concentrations  hi  gelatine, 
agar-gelatine,  and  agar-protein. 

The  amine  of  this  group  was  now  tested  both  in  solution  and  in- 
corporated in  agar  sections.  Plates  of  agar  (9  parts)  and  asparagine 
(1  part)  were  prepared  and  swelled  in  comparison  with  aspartic  acid, 
giving  results  shown  in  table  47. 


Effect  of  Certain  Organic  Acids  and  Amino  Compounds. 
TABLE  47.— 16°  to  18°  C. 


59 


Citric 

Sodium 

Water. 

acid, 

hydroxid, 

0.01  N. 

0.01  M. 

p.  ct. 

p.  ct. 

p.  ct. 

Agar  9,  asparagine  1  

640 

300 

402 

Agar  9,  aspartic  acid  1  .... 

296 

250 

625 

The  proportion  of  the  acid  and  the  asparagine  being  too  high 
to  be  of  any  physiological  interest,  new  plates  with  half  the 
quantity  of  acid  and  amine  were  prepared,  and  these  came  down 
to  a  thickness  of  0.32  and  0.33  mm.,  which  swelled  1,875  per  cent 
in  distilled  water  as  compared  with  agar,  which  showed  a  hydration 
capacity  of  2,700  per  cent.  The  effect  in  this  trial  was  not  so 
marked  as  in  the  first  series,  but  it  is  evident  that  the  incorpora- 
tion of  the  asparagine  in  any  proportion  in  the  colloid  affects 
hydration  to  a  greater  extent  than  the  perfusion  of  the  asparagin 
in  the  same  concentration,  which  in  this  case  gave  swellings  of  2,300 
per  cent.  Even  a  0.1  M  solution  with  double  the  amount  present  in 
the  solution  did  not  reduce  the  hydration  to  the  limits  shown  by  the 
agar-asparagine  plate  used  in  this  test.  The  asparagine  was  present  in 
such  amount  that  if  diffused  out  of  the  sections  it  would  have  made  a 
0.04  M  solution  in  the  30  c.c.  of  water  in  the  dish. 

Asparagine  was  now  applied  in  a  series  of  concentrations  to  sections 
of  agar  of  the  above  swelling  capacity  in  water  and  it  was  found  that 
hydration  was  actually  increased  or  accelerated  by  the  presence  of  the 
amine.  That  this  result  did  not  simply  appear  by  faulty  comparisons 
was  shown  by  the  following  replacement  test: 

A  trio  of  sections  which  had  been  swelled  in  distilled  water  to  a  total 
of  2,630  per  cent,  and  which  had  stood  in  the  solution  without  any 
perceptible  change  for  a  few  hours  after  the  close  of  the  test,  was  now 
treated  with  a  0.01  M  asparagine  solution.  The  mechanical  disturbance 
which  might  result  from  changing  the  liquid  in  the  dishes  was  mini- 
mized by  fractionization.  About  one-third  of  the  water  was  removed, 
the  level  was  raised  by  the  addition  of  asparagine  solution,  and  this 
was  repeated  about  a  half-dozen  tunes,  the  final  result  being  a  solution 
which  was  diluted  slightly  below  the  hundredth  normal.  A  slow 
expansion  began  at  once,  which  continued  for  about  20  hours,  which 
raised  the  total  hydration  of  these  sections  to  2,890  per  cent,  an  in- 
crease of  230  per  cent,  due  to  the  action  of  the  asparagine  on  sections 
which  had  undoubtedly  been  reduced  in  mass  somewhat  by  solution 
from  the  surfaces. 

When  asparagine  is  applied  to  mixtures  in  which  the  gelatine  is 
replaced  by  an  albumin,  the  results  included  some  special  reactions. 
Plates  of  agar  and  oat-protein  were  made  up  to  contain  8  parts  of  the 


60  Hydration  and  Growth. 

first  and  2  of  the  last,  coming  down  to  a  thickness  of  0.22  to  0.23  mm. 
These  swelled  at  17°  C.  to  the  proportions  shown  in  table  45,  which  in 
some  cases  exceeded  that  in  water.  The  swelling  in  concentrations  as 
high  as  0.01  M  were  but  little  below  that  in  water. 

Glycocoll  has  been  used  in  many  cultural  tests  with  plants  and 
various  interpretations  have  been  placed  on  its  accelerative  influence 
on  growth.  The  experiments  with  this  material,  therefore,  included 
the  possibilities  of  the  manner  and  extent  to  which  this  might  accom- 
pany or  run  parallel  with  hydration  reactions. 

The  first  trials  were  made  with  this  reagent  incorporated  with 
liquid  agar  in  such  proportion  that  the  amount  present  in  three  sec- 
tions would  have  been  equivalent  to  that  in  30  c.c.  of  0.14  M  solution. 
Trios  of  such  sections  0.15  mm.  thick  gave  swellings  of  1,133,  1,267 
and  1,300  per  cent  hi  water  at  16°  C.,  which  is  much  less  than  that 
shown  in  a  solution  at  0.3  M  containing  twice  as  much  of  the  amino- 
acid.  (Table  45). 

Thin  sections  of  agar  swelled  in  all  glycocoll  solutions  less  concen- 
trated than  0.3  M  to  the  amplitude  attained  in  water  and  exceeded  it  in 
some  cases,  a  fact  which  for  the  first  time  gives  a  sound  basis  for  cul- 
tural tests  in  which  growth  was  accelerated  and  the  total  increased  by 
this  compound. 

Another  pentosan,  gum  tragacanth,  was  dried  from  solutions  to  form 
sections  0.13  mm.  thick  on  filter-paper.  Swellings  at  15°  C.  were  ob- 
tained, as  shown  in  table  48. 

TABLE  48.  p.  ct. 

Distilled  water 1 ,380 

Glycocoll,  0.03M 1,382 

Glycocoll,  0.05M •< 1,077 

Glycocoll,  0.01  M 1,462 

This  gum  liquefies  irregularly,  and  hence  the  figures  show  the  extent 
of  swelling  before  active  dispersion  of  the  mass  begins. 

A  mixture  of  9  parts  gelatine  and  1  part  gum  tragacanth  was  made 
up  at  25  per  cent  to  correspond  to  a  similar  mixture  of  gelatine  and 
opuntia  mucilage.  Swellings  as  follows  at  15°C.  were  obtained: 

TABLE  49.  p.  ct. 

Distilled  water 1,320 

Glycocoll,  3  M 1,520 

Glycocoll,  0.05M 1,040 

Glycocoll,  0.01  M 1,320 

Nothing  may  be  concluded  on  the  basis  of  these  figures,  except  that 
the  hydration  of  this  material  reaches  a  stage  where  it  goes  into  dis- 
persion unevenly  and  in  a  manner  which  makes  auxographic  readings, 
as  well  as  all  mass  or  weight  determination,  of  doubtful  value. 

The  above  tests  were  repeated  with  opuntia  mucilage  at  15°  C., 
with  results  as  shown  in  table  50. 


Effect  of  Certain  Organic  Adds  and  Amino  Compounds.  61 

TABLE  50.  p.  d. 

Distilled  water 923 

Glycocoll  3M 800 

Glycocoll  0.05  M 664 

Glycocoll  0.01  M 600 

Here  again  the  uneven  dispersion  of  the  mucilage  results  in  auxo- 
graphic  records,  the  obvious  meaning  of  which  would  be  unsafe  to 
follow.  It  is  highly  probable  that  the  high  relative  swelling  in  the 
concentrated  solution  is  due  to  coagulatory  or  aggregation  effects, 
especially  on  the  surfaces  of  the  sections,  resulting  in  a  sac-like  con- 
dition which  would  show  considerable  increase  before  dispersion 
began,  resulting  in  a  final  shrinkage.  This  dispersion  began  earlier 
in  the  weaker  solutions. 

Swellings  of  gelatine  in  glycocoll  ran  uniformly  low,  the  presence  of 
this  substance  apparently  accelerating  solution  of  the  gel. 

Sections  consisting  of  4  parts  agar  and  1  of  gelatine  which  had  an 
average  thickness  of  0.3  mm.  swelled  as  follows  at  15°  C.  in  glycocoll: 

TABLE  51.  p.  a. 

Glycocoll,  0.3M 1,550 

Glycocoll,  0.05M 1,233 

Glycocoll,  0.01  M 1,960 

Glycocoll,  0.002  M 1,767 

The  average  swelling  of  such  sections  in  water  was  about  1,700  per 
cent  and  the  irregularity  characteristic  of  auxographic  measurements 
of  the  action  of  this  amino-acid  is  seen  in  the  above  results. 

A  preparation  was  now  made  in  which  2  parts  of  the  water-soluble 
protein  from  oats  was  added  to  8  parts  of  agar  in  a  2.5  per  cent  solution 
of  the  latter.  The  plates  dried  to  a  thickness  of  0.25  mm.  When 
sections  of  such  biocolloids  were  swelled  in  the  glycocoll  series,  the 
results  were  as  shown  in  table  45,  the  hydration  in  concentrations  less 
than  0.01  M  approaching  and  surpassing  those  in  distilled  water. 

A  number  of  tests  were  made  to  determine  the  influence  of  glycocoll 
on  hydrations  in  acetic  acid.  The  first  was  that  of  surface  slices  of 
Opuntia,  which  had  dried  to  a  thickness  of  0.8  mm.  Trios  swelled  163 
per  cent  in  0.05  N  acetic  acid  and  156  per  cent  in  a  0.05  N  solution 
of  acetic  acid  and  glycocoll  each.  No  especial  significance  can  be 
attached  to  the  lesser  swelling  in  the  double  solution,  except  that  no 
evidence  as  to  acceleration  of  swelling  by  the  addition  of  the  amino-acid 
was  obtained. 

Next,  trios  of  sections  of  8  parts  agar  and  2  parts  gelatine  0.3  mm.  in 
thickness  were  swelled  hi  the  acetic  and  amino-acetic  solutions  0.01  N 
at  18°  C.  The  swelling  in  the  acetic  acid  alone  was  1,450  per  cent, 
while  that  in  the  combined  solutions  was  but  1,300  per  cent,  which 
agreed  with  the  previous  effects  in  being  less  than  in  the  acid  alone. 
It  is  to  be  noted  that  the  amount  of  the  acetic  acid  in  the  combined 
solution  in  the  swelling-dish  would  be  but  half  that  when  this  acid  was 
used  alone. 


62  Hydration  and  Growth. 

Trios  of  sections  of  agar  swelled  1,875  per  cent  in  a  0.01  N  solution  of 
acetic  acid  at  18°  C.,  while  a  combined  solution  of  equivalent  molecular 
concentration  showed  a  swelling  of  1,750  per  cent. 

There  now  remained  the  test  with  living  tissues.  Some  joints  of 
Opuntia  blakeana  of  1918,  which  had  been  brought  from  Tucson  two 
months  earlier  and  had  laid  on  the  table,  with  the  result  that  they  had 
lost  much  water  but  were  still  alive,  were  used  for  this  test.  A  trio  of 
sections  with  an  average  thickness  of  6  mm.  swelled  60  per  cent  in  the 
hundredth-normal  acetic  acid,  while  a  similar  trio  which  measured 
5.5  mm.  on  the  average  swelled  but  45.5  per  cent  in  the  combined 
acetic-glycocoll  solution.  A  second  feature  distinguished  the  two 
reactions,  the  swelling  in  the  acetic  acid  being  continuous  and  ap- 
proaching zero  during  the  20  hours  of  measurement,  while  in  the  com- 
bined solution  full  expansion  was  reached  in  4  hours,  after  which  a 
shrinkage  resulted  in  a  loss  of  nearly  5  per  cent,  suggesting  that  the 
H-ion  concentration  of  the  combined  solution  was  greater  than  that 
of  the  acid  alone. 

A  return  was  made  to  the  biocolloidal  mixtures  and  trios  of  sections 
of  agar  8  parts  and  oat  protein  2  parts,  with  a  thickness  of  0.22  mm., 
swelled  at  18°  C.  The  hydration  in  the  hundredth-normal  acetic  acid 
gave  an  increase  of  1,318  per  cent,  while  an  equimolecular  solution  of 
the  acetic  acid  and  glycocoll  gave  a  swelling  of  1,605  per  cent.  This 
test  is  the  only  one  of  the  series  in  which  the  addition  of  glycocoll  to 
the  acetic  acid  enhances  imbibition.  In  this  last  test  the  amount  of 
solution  poured  in  each  dish  was  such  that  the  same  quantity  of  the 
acetic  acid  was  present  in  both. 

An  additional  test  was  made  in  which  equal  amounts  of  glycocoll  and 
acetic  acid  were  brought  together  at  a  concentration  of  0.001  M  each 
on  agar-oat  protein  sections  as  above.  The  swelling  in  the  acetic 
acid  was  2,681  per  cent,  or  about  the  same  of  that  possible  in  distilled 
water  (2,630  per  cent),  while  the  swelling  in  the  combined  solution  was 
slightly  less,  being  2,570  per  cent. 

Glycocoll  and  other  amino-groups  are  present  in  the  plant  in  com- 
paratively great  dilutions,  and  probably  at  no  tune  does  the  amount 
present  reach  the  concentration  in  which  a  retardation  or  limiting  of 
the  hydration  effect  would  be  exerted.  The  experiments  described 
show  that  glycocoll  and  asparagin  may  actually  increase  the  hydration 
capacity  of  pentosan  and  of  pentosan-protein  colloids.  The  meager 
results  obtained  from  swelling  plant-sections  are  not  harmonious  and 
further  experimentation  is  highly  desirable.  The  accelerating  effect 
of  glycocoll  is  a  subject  which  has  come  up  for  notice  many  times. 
Dakin  connected  its  action  with  possible  catalytic  effects.1  The  simi- 
larity of  the  results  obtained  from  agar  and  agar-protein  mixtures 

1  Dakin,  H.  D.     The  catalytic  action  of  amino-acids,  etc.,  in  effecting  certain  syntheses.     Jour. 
Biol.  Chem.,  7:  49-55.     1909. 


Effect  of  Certain  Organic  Adds  and  Amino  Compounds.  63 

and  from  the  swelling  of  plants  makes  it  fairly  certain  that  the  effect 
is  due  primarily  to  the  action  of  the  pentosaus. 

The  most  recent  tests  of  the  effects  of  glycocoll  on  plants  are  those 
of  Borowikow,1  completed  in  1913  and  published  in  the  same  year,  and 
those  of  Dachnowski,  brought  out  in  1914.2  Borowikow  took  the 
position  that  substances  which  facilitate  hydration  of  the  plasmatic 
colloids  accelerate  growth,  and  that  such  hydration  was  one  of  pro- 
teins, an  assumption  which  is  not  sound.  His  trials  consisted  in  com- 
paring the  growth  of  seedlings  of  Helianthus  in  distilled  water  as  a 
check  or  control  with  the  substances  to  be  tested  added  to  water,  the 
measurements  being  taken  during  a  few  hours  only.  Glycocoll  was 
used  in  0.01  N  and  0.005  N  concentration.  Such  concentrations  are 
relatively  high  for  the  plant,  and  only  retardation  effects  were  obtained. 

Dachnowski's  figures  indicate  that  glycocoll  added  to  hydrochloric 
acid  in  concentrations  of  N/1,600  (50  c.c.  N/800  of  each  substance) 
causes  an  increase  in  the  amount  of  water  absorbed  by  bean  seeds, 
and  a  lesser  increase  of  hydration  in  corn  seeds. 

Both  absorption  and  transpiration  by  cuttings  of  tomato  were  less 
in  solutions  of  hydrochloric  acid  ranging  from  N/800  to  N/6,400  than 
in  water,  but  this  retarding  effect  was  counteracted  to  some  extent 
when  glycocoll  was  added  to  the  solutions.  This  amino-acid  also 
caused  an  increased  gain  in  weight  in  acid  and  alkaline  solutions. 

The  hydration  phenomena  described  in  the  preceding  pages  afford 
some  interesting  parallelisms  with  the  action  of  these  compounds  on 
growth,  absorption,  and  transpiration. 

It  is  evident  that  we  must  definitely  and  finally  cease  to  treat  a  plant 
cell-mass  as  an  amphoteric  colloid  with  a  dissociation  expressed  by  the 
actual  acidity  of  the  cell-sap.  Such  dissociation  and  resultant  hy- 
dration capacity  may  determine  the  action  of  protoplasts  or  of  cell- 
organs  which  are  chiefly  proteinaceous. 

Vegetative  cell-masses  such  as  are  responsible  for  growth,  and  the 
activity  of  which  constitutes  growth  in  the  larger  sense,  are  composed 
of  colloids  predominantly  of  a  carbohydrate  character.  These  pen- 
tosans  do  not  dissociate.  Their  swelling  capacity  in  electrolytes  is 
less  than  in  pure  water.  The  hydration  of  agar  and  the  pentosans  in 
acids  is  retarded  or  lessened  by  the  action  of  H  ions,  so  directly  that 
the  proportionate  swelling  of  agar  in  an  acid  such  as  acetic  or  succinic 
might  be  used  as  a  measure  of  the  concentration  of  the  acid  solution 
(see  p.  57).  This  fact  and  the  part  played  by  the  dissociation  of 
gelatine  may  be  traced  through  all  of  the  results  on  hydration  of  agar, 
agar-gelatine,  and  agar-protein  mixtures.  Thus,  for  example,  agar 

1  Borowikow,  G.  A.    Ueber  die  Ursachen  des  Wachstums  der  Pflanzen.    Biochem.  Zeitschrift, 
50:  119.     1913. 

2  Dachnowski,  A.     The  effects  of  acid  and  alkaline  solutions  upon  the  water-relations  and  the 
metabolism  of  plants.     Amer.  Jour,  of  Bot.,  1:  412-439.     1914;  also,  Dachnowski  and  Gormley. 
The  physiological  water  requirement  and  the  growth  of  plants  in  glycocoll  solutions.     Amer. 
Jour,  of  Bot.,  1 :  174-185.     1914. 


64  Hydration  and  Growth. 

alone  gives  an  average  swelling  of  about  2,600  per  cent  of  plates  0.18 
to  0.20  mm.  in  thickness  at  13°  C.  When  combined  with  gelatine  in 
proportions  of  8  to  2,  the  swelling  is  less  than  1,700  per  cent. 

The  reactions  of  the  pentosans  and  pentosan-protein  colloids  in 
solutions  of  the  ammo-compounds  show  some  highly  important  depart- 
ures, the  chief  of  which  is  the  fact  that  the  hydration  capacity  is 
greater  than  in  distilled  water  in  such  monobasic  acids,  but  not  in  the 
dibasic  aspartic  acid.  This  last-named  substance  dissociates,  so  that 
.01  M,  pH  =  3,  in  accordance  with  which  it  is  found  to  lessen  the 
hydration  capacity  of  agar,  but,  on  the  other  hand,  this  action  is  not 
shown  by  the  pentosan-protein  mixture.  No  explanation  may  be 
offered  for  this  behavior  and  for  the  excessive  swelling  of  pentosans 
and  pentosan  mixtures  in  amino- compounds,  except  that  amino-com- 
pounds  may  form  salts  with  the  carbohydrate,  thus  increasing  the 
hydration  capacity  of  the  latter.  That  this  superior  swelling  is  an 
actuality  is  well  demonstrated  by  the  increase  that  resulted  when  agar- 
albumin  sections  in  a  condition  approaching  complete  hydration  showed 
a  further  marked  increase  when  the  water  was  replaced  with  an 
asparagin  solution.  The  positive  action  of  the  ammo-compounds  is 
also  well  demonstrated  by  the  fact  that  the  maximum  effects  were 
produced  at  a  concentration  not  coincident  with  the  maximum  con- 
centration and  at  a  point  of  great  dilution.1 

When  these  results  are  applied  to  the  conditions  in  the  cell,  emphasis 
is  given  to  the  fact  that  the  total  of  amino-acids  is  always  no  more  than 
a  fraction  of  the  amount  of  organic  acids  present.  It  is  highly  probable 
that  these  substances,  originating  constructively  in  the  plant  and 
affecting  growth  in  a  profound  manner,  may  do  so  partly  by  their 
participation  in  the  buffer  processes. 

1  MacDougal  and  Spoehr.     The  effect  of  organic  acids  and  their  ammo-compounds   on  the 
hydration  of  agar  and  on  a  biocolloid.     Proc.  Soc.  Exper.  Biol.  and  Med.,  16:  33.     1918. 


VI.  REACTIONS  OF  BIOCOLLOIDS  AND  CELL-MASSES  TO 

CULTURE  SOLUTIONS,  BOG,  SWAMP,  AND  GROUND 

WATER,  AND  OTHER  SOLUTIONS. 

The  organism  encounters  a  variety  of  substances  in  solution  in  the 
substratum  or  medium  to  which,  of  course,  the  colloids  of  the  cell  re- 
act in  a  manner  determined  by  their  own  composition  and  that  of  the 
impinging  substances.  The  securest  knowledge  of  the  complex  rela- 
tions involved  will  in  the  end  rest  upon  results  obtained  by  analytical 
experiments  in  which  the  effects  of  separate  substances  and  graded 
concentrations  of  the  elements  are  first  determined  and  then  their 
action  in  combination  is  measured.  Meanwhile,  a  number  of  standard 
or  commonly  accepted  solutions  are  used  for  a  variety  of  cultural  and 
experimental  purposes  and  an  effort  was  made  to  ascertain  the  reactions 
of  biocolloids  and  of  sections  of  plants  to  them  in  terms  of  imbibitional 
swelling.  The  idea  was  extended  to  include  the  "natural  waters" 
which  are  characteristic  of  some  well-defined  plant  habitats,  such  as 
bogs  and  swamps. 

A  large  and  important  share  of  the  knowledge  of  the  physiology  of 
plants  rests  upon  cultures  made  with  "  nutrient  solutions."  One  of 
these,  after  a  formula  devised  by  W.  E.  Tottingham,  was  chosen  for 
the  test.1  Its  composition  was :  potassium  nitrate  4.048  g.,  dipotassic 
phosphate  12.980  g.,  magnesium  sulphate  crystals  29.280  g.,  and  cal- 
cium nitrate  27.920  g.,  in  4,000  c.c.  of  water.  A  precipitate  comes 
down  in  the  bottle  on  standing.  This  was  filtered  out  and  dissolved 
in  distilled  water,  which  was  used  to  dilute  the  solution  to  a  concen- 
tration of  about  0.5  per  cent  total  concentration. 

The  preliminary  trial  of  the  effect  of  the  whole  solution  was  made 
with  sections  of  a  plate  consisting  of  95  parts  agar  and  5  parts  of  bean 
protein,  an  old  preparation  which  had  been  exposed  to  the  damp  air 
for  a  month.  The  swelling  measurements  were  as  follows: 

TABLE  52. 

p.  ct. 

Water 617.6 

Nutrient  solution,  0.5  p.  ct 500 

Citric  acid,  O.Ql  N 406.8 

Sodium  hydroxid,  0.01  M 431.4 

The  only  feature  of  interest  hi  the  results  was  the  low  imbibition  in 
water,  the  dried  sheet  being  an  old  one.  A  fresh  preparation  was 
made  with  the  agar  and  bean  protein  hi  the  same  proportion  as  before, 
and  a  double  series  of  instruments  was  used  in  the  test. 

In  order  to  determine  the  possible  interference  or  antagonism  of  the 
constituents,  tests  were  also  made  of  the  separate  action  of  the  four 

1  Tottingham,  W.  E.    A  quantitative  chemical  and  physiological  study  of  nutrient  solutions  for 
plant  cultures.     Physiol.  Res.,  1 :  No.  4.     May  1914. 

65 


66 


Hydration  and  Growth. 


salts  in  the  concentrations  in  which  they  occur  in  the  culture  solution 
(table  53). 

The  total  amount  of  swelling  in  the  culture  solution  is  scarcely 
more  than  half  that  in  distilled  water,  that  in  the  dipotassic  phosphate 
not  falling  much  below  that  of  water.  Swelling  in  potassium  nitrate 
is  much  greater  than  that  shown  in  the  culture  solution.  The  low 
imbibition  in  calcium  nitrate  is  in  accordance  with  the  expectancies. 

TABLE  53. 


Dist. 
water. 

Nutrient 
soln., 
0.5  per  ct. 

Potass, 
nitrate, 
M  0.00285. 

Magn. 
sulphate, 
M  0.00898. 

Calc. 
nitrate, 
M  0.00844. 

Di-potass. 
phosphate, 
M  0.00681. 

Swelling  in  10  hours  

p.  ct. 
1,360 
1,300 

p.  ct. 
720 
760 

p.  ct. 
1,040 
900 

p.  ct. 
740 
740 

p.  ct. 
340 
600 

p.  ct. 
1,200 
940 

Average  

1,330 

750 

970 

740 

470 

1,070 

Swelling  in  24  hours  .... 

1,560 

800 

1,260 

804 
800 

680 
500 

1,400 
1,300 

Average  

1,560 

800 

1,260 

802 

590 

1,350 

The  magnesium  salt  exercises  an  imbibitional  action  equivalent  to 
that  of  the  complete  solution.  The  potassium  salts  allow  a  notably 
greater  swelling.  Apparently  the  calcium  salt  interferes,  or  exercises 
an  antagonism  which  results  in  the  averaged  total  exemplified.  The 
actual  relative  action  of  these  salts,  however,  can  not  be  taken  up  at 
this  time.  A  consideration  of  the  imbibitional  action  of  the  constit- 
uent salts  might  yield  some  data  which  would  be  of  value  in  deter- 
mining the  composition  of  culture  solutions  for  special  purposes. 

A  similar  series  of  tests  of  the 

effect  of  the  nutrient  solution  TABLE  54. 

and  its  components  upon  grow- 
ing tissues  were  made  with 
sections  of  young  stems  of 
Rudbeckia  bearing  young  flower- 
heads.  Tangential  slices  were 
removed  from  one  side  to  allow 
expansion  and  trios  of  pieces  a 
centimeter  long  and  3.5  mm.  in 
thickness  were  placed  under  the 
auxographs  in  a  dark  room  at 

16°  C.  Air-dried  sections  of  the  same  stems  which  had  been  exposed 
to  the  air  and  light  for  a  day  and  had  shrunk  to  about  half  of  their 
original  thickness  were  now  placed  in  identical  solutions.  The  swell- 
ings of  the  fresh  and  of  the  dried  sections  are  given  in  table  54. 


Living 
sections. 

Dried 
sections. 

Water  

p.  ct. 
5.8 

p.  ct. 
28  6 

Nutrient  solution  

4 

15  7 

Potassium  nitrate  

4.3 

27.1 

Magn.  sulphate  

2.8 

20 

Calcium  nitrate  

2 

3.6 

Di-potassium  phosphate  .  . 

2.9 

12.8 

Certain  Reactions  of  Biocolloids  and  Cell-masses.  67 

Several  variations  are  apparent,  but  perhaps  the  relations  of  greatest 
importance  are  those  which  may  be  expressed  by  saying  that  the 
imbibition  of  dried  specimens  is  five  times  that  of  living  material  in 
distilled  water,  only  four  times  in  nutrient  solution,  over  six  times  in 
potassium  nitrate,  over  seven  times  in  magnesium  sulphate,  less  than 
twice  in  calcium  nitrate,  and  over  four  times  in  dipotassium  phos- 
phate. It  is  to  be  noted  that  when  a  section  of  living  tissue  is  dehy- 
drated it  is  not  possible  to  restore  the  cell-colloids  to  their  original 
condition  simply  by  swelling.  This  is  due  chiefly  to  the  fact  that,  as 
desiccation  proceeds,  the  salts,  acids,  sugars,  etc.,  in  the  liquids  are 
concentrated  until  finally  they  are  fixed  by  the  solidifying  proto- 
plasmic gel  in  this  condition.  Rehydration  must  then  take  place  as 
in  a  salted  colloid  with  the  sugars  in  a  concentration  in  which  they 
may  modify  imbibition. 

Petioles  of  young  leaves  of  Phytolacca,  with  a  thickness  of  3  mm., 
swelled  4.2  per  cent  in  distilled  water  and  an  equal  amount  in  potassium 
phosphate,  5  per  cent  in  magnesium  sulphate,  3.3  per  cent  in  calcium 
nitrate,  and  -2.5  per  cent  in  the  nutrient  solution.  The  equivalence 
or  uniformity  of  the  material  was  in  doubt,  however,  and  the  test  was 
not  extended  to  dried  sections. 

The  4-angled  stems  of  Mentha  spicata  offered  certain  mechanical 
advantages,  and  the  internodes  near  the  apex  of  the  stem  which  were 
half-grown  and  with  a  thickness  of  3  mm.  were  selected.  Trios  of 
sections  3  or  4  mm.  long  were  tested  with  distilled  water,  culture 
solution,  and  its  components.  The  swelling  of  fresh  specimens  was 
very  slight,  varying  from  0.05  to  0.1  mm.,  and  no  safe  comparisons 
could  be  made.  Dried  sections  came  down  nearly  half  of  their  original 
dimensions,  and  when  a  series  was  swelled  in  distilled  water  the  in- 
crease was  but  12.9  per  cent  and  in  hundredth-normal  citric  acid  6.4 
per  cent,  while  in  hundredth-molar  sodium  hydroxid  the  increase  was 
19.3  per  cent.  These  results  indicate  that  the  plant  colloids  were  in 
an  acidified  condition,  the  swelling  in  water  and  hi  acid  being  conse- 
quently small,  while  that  in  alkali  was  a  swelling  in  a  neutralized  or 
nearly  neutralized  condition.  The  dried  series  in  the  culture  solution 
and  its  components  gave  swellings  of  16.8  per  cent  in  distilled  water, 
almost  no  swellings  in  nutrient  solution  (due  to  defective  wetting 
by  the  liquid),  19.3  per  cent  in  potassium  nitrate,  12.6  per  cent  in 
magnesium  sulphate,  9.6  per  cent  in  calcium  nitrate,  and  4.8  per  cent 
in  dipotassic  phosphate.  The  swelling  of  dried  sections  presents  some 
possible  sources  of  error  in  the  limited  surfaces  presented  for  absorption 
which  may  be  "'waterproofed"  in  some  cases,  while  in  other  instances 
the  sections  collapse  or  do  not  swell  toward  their  original  form. 

Direct  effects  of  the  waters  of  bogs  and  swamps  in  producing  modi- 
fications of  growth,  departures  in  structure  and  form,  and  in  influenc- 
ing general  nutrition  are  well  established  and  have  long  been  known. 


68  Hydration  and  Growth. 

The  numerous  analyses  of  the  water  have  failed  to  disclose  physico- 
chemical  features  which  might  be  held  responsible  for  the  very  direct 
and  positive  action  exercised  in  the  determination  of  the  plant  form- 
ations of  such  places.  The  history  of  such  attempts  is  a  long  one.1 
Bog  water  was  furnished  by  Mr.  E.  R.  Long,  who  procured  a  sample 
from  Ronalds,  in  the  region  of  Seattle,  Washington.  Dr.  J.  M.  McGee 
reports  the  following  constituents  per  liter: 

TABLE  55.  gm. 

Organic  matter 0 . 106 

Ash  (chiefly  CaSO«) 048 

Total  soluble  residue 154 

Swamp  water  was  procured  by  the  kindness  of  Dr.  S.  A.  Gortner,  who 
obtained  a  sample  from  near  Anoka,  Minnesota,  concerning  which  he 
says: 

"This  sample  was  taken  about  20  miles  north  of  Minneapolis,  in  Anoka 
County,  of  which  three-fourths  of  the  area  consists  of  peat  lands.  These 
peat  lands  are  of  the  grass  and  sedge  formation,  the  peat  being  from  4  to  6 
inches  or  more  deep,  fairly  well  decomposed,  and  one  of  the  better  grades  of 
peat  for  agricultural  purposes  in  that  it  contains  an  appreciable  amount  of 
lime.  I  believe  that  you  will  find  this  sample  of  water  perfectly  typical  of 
most  of  the  large  grass  bogs  of  Minnesota." 

The  analysis  of  this  water  shows  the  following  per  liter: 

TABLE  56.  gm. 

Soluble  organic  matter 0.094 

Ash  (CaSO4  with  trace  of  NaCl) 128 

Total  soluble  residue 222 

The  degree  of  acidity  of  the  bog  water  was  such  that  1.1  c.c.  of  N/10 
potassium  hydrate  was  necessary  to  neutralize  100  c.c.  of  water.  The 
acidity  of  the  grass-sedge  water  was  scarcely  a  third  of  this,  but  0.35 
c.c.  N/10  potassium  hydrate  being  necessary  to  neutralize  100  c.c. 

The  first  trial  of  the  action  of  these  waters  and  comparative  solutions 
was  made  with  living  material.  Circular  disks  12  mm.  across  and  of 
an  average  thickness  of  11  to  13  mm.  were  cut  from  joints  of  Opuntia 
discata  which  had  matured  at  Carmel,  California,  in  the  summer  of 
1917.  Tests  were  made  with  water,  swamp  water,  bog  water,  and 
various  calcium  solutions  at  15°  C.  The  measurements  obtained  were 

as  follows: 

TABLE  57.  p.  ct. 

Distilled  water 18.2 

Swamp  water 13.6 

Bog  water 18.3 

Calcium  nitrate,  0.008M 15.5 

Calcium  nitrate,  0.008  M  acidified  with,  nitric  acid 16.8 

Calcium  nitrate,  2  M  (shrinkage  and  subsequent  swelling) '  6.8 

Calcium  nitrate,  0.2  M  (steady  shrinkage) 

Calcium  nitrate,  0.02M 14.6 

Calcium  nitrate,  0.002M 15.5 

Calcium  nitrate,  0.0002  M 21 

1  See  Bigg,  G.  B.,  Summary  of  bog  theories.    Plant  World,  19:310,  1916. 


Certain  Reactions  of  Biocolloids  and  Cell-masses.  69 

The  uppermost  line  shows  a  swelling  in  bog  water  equivalent  to 
that  in  distilled  water,  while  the  imbibition  in  swamp  water  is  very 
much  less,  sustaining  about  the  same  proportions  as  the  measurements 
of  the  swelling  of  biocolloids.  The  retarding  action  of  the  swamp 
water,  high  in  calcium,  is  greater  than  that  of  the  solution  0.008  M, 
in  which  this  salt  enters  into  nutrient  solutions,  and  greater  even  than 
that  of  a  0.02  M  solution.  The  retarding  action  of  swamp  water  may 
be  predicted  to  be  about  the  same  as  a  0.03  M  solution  of  calcium  nitrate 
acidified  as  in  the  solution  used.  Such  acidification  was  made  by  adding 

1  c.c.  of  hundredth-molar  nitric  acid  to  25  c.c.  of  the  calcium  solution. 
The  final  cessation  of  shrinkage  and  slight  enlargement  of  sections 

in  the  2  M  solution  remains  unexplained,  since  the  shrinkage  in  a 
solution  containing  but  one-tenth  of  this  amount  of  calcium  was  con- 
stant during  the  entire  90  hours  of  the  exposure.  The  final  figures  in 

2  M  to  0.0002  M  are  of  swellings  which  were  continued  for  90  hours, 
while  the  swellings  in  water,  swamp  water,  bog  water,  calcium  as  in  a 
nutrient  solution,  and  acidified  calcium  solution  were  taken  at  the  close 
of  40  hours.     The  total  swelling  of  21  per  cent  in  calcium  nitrate 
0.0002  M  is  probably  equivalent  to  that  of  distilled  water.     The  swell- 
ings of  biocolloids  in  the  same  solutions  should  receive  attention  in  this 
connection.1 

In  addition  to  the  above  note  on  the  swelling  of  the  sections  in  the 
2  M  solution  of  calcium  nitrate,  the  f ollowing  facts  are  of  interest : 
Four  sections  with  an  aggregate  thickness  of  37  mm.  were  placed  in  a 
dish  and  covered  with  such  a  solution  at  the  time  the  auxograph 
measurements  were  started.  As  the  sections  under  the  auxograph 
were  in  an  expanding  stage  when  the  measurements  were  closed,  free 
sections  were  allowed  to  remain  in  the  dish  after  the  records  on  the 
instrument  were  ended.  The  free  sections  were  measured  6  days  after 
being  put  in  the  concentrated  solution,  with  the  result  that  their  total 
thickness  was  found  to  be  42  mm.,  a  gain  of  5  mm.  or  13.3  per  cent. 

Sections  of  a  biocolloid  consisting  of  agar  90  parts  and  oat  protein 
10  parts  were  swelled  in  a  series  of  solutions  of  calcium  nitrate  parallel 
to  the  above  set.  The  increase  in  the  0.5  M  solution  was  975  per  cent, 
525  per  cent  in  the  0.2  M  solution,  650  per  cent  in  the  0.02  M  solution, 
1,425  per  cent  in  the  0.002  M  solution,  and  1,975  per  cent  in  the 
0.0002  M  solution.  The  test  was  repeated  with  the  following  results 
at  the  end  of  24  hours:  swelling  in  2  M  solution,  917  per  cent;  in  0.2  M 
solution,  722  per  cent;  in  0.02  M  solution,  777  per  cent;  in  0.002  M 
solution,  1,555  per  cent. 

The  minimum  swelling  in  this  series  evidently  lies  between  the  con- 
centrations of  0.2  M  and  a  molar  solution. 

Another  series  was  carried  out  in  which  sections  of  Opuntia  were 
swelled  at  temperatures  of  18°  to  20°  C.  in  acidified  and  salt  solutions, 
as  given  in  table  58. 

1  MacDougal,  D.  T.     The  effect  of  bog  and  swamp  waters  on  swelling  in  plants  and  in  biocol- 
loids.    Plant  World,  21:88.     1918. 


70  .  Hydration  and  Growth. 

TABLE  58. 

p.  ct. 

Swamp  water 14 

Swamp  water,  citric  acid,  0.01  N 15 

Potassium  nitrate,  0.01  M 14 

Citric  acid,  0.01  N '.'.'.'.'.  7 

Potassium  nitrate,  citric  acid,  0.01  N 9 

Potassium  hydroxid,  0.01  M 12 

The  measurements  in  swamp  water  alone  and  with  acid  include  the 
full  increase  in  96  hours,  while  the  others  extended  over  from  20  to 
40  hours. 

No  important  effect  can  be  ascribed  to  the  acidification  of  swamp 
water.  The  swelling  of  the  sections  in  the  hundredth-molar  solution 
of  potassium  nitrate  was  but  little  below  that  in  the  swamp  water,  but 
when  this  solution  was  similarly  and  equally  acidified,  a  decrease 
ensued.  The  foregoing  tests  were  made  with  sections  in  a  living  con- 
dition, in  which  questions  of  permeability  and  osmotic  action  might 
possibly  play  a  part.  Material  was  prepared  to  exclude  the  action  of 
the  living  cell.  The  chlorophyllous  layers  were  removed  from  the  two 
sides  of  joints  of  Opuntia  and  slices  7  mm.  in  thickness  were  cut  in  the 
plane  of  the  joint  and  placed  between  two  sheets  of  filter-paper,  to 
one  of  which  they  adhered.  A  third  sheet  was  laid  over  them  and 
the  preparation  placed  on  a  wire  netting  to  dry  without  pressure.  In 
6  days  the  thickness  had  been  reduced  to  about  0.5  mm.  and  enough 
moisture  still  remained  to  give  the  slices  a  leathery  consistency.  Suit- 
able sections  free  from  visible  fibrovascular  tissue  were  prepared  which 

gave  swellings  as  follows: 

TABLE  59. 

Living  sections.     Dried  sections, 

p.  ct.  p.  ct. 

Distilled  water 660  47 

Bog  water 640  45 

Swamp  water 530  38 

Culture  solution,  0.5  per  cent 625  44 

The  measurements  were  taken  at  the  end  of  24  hours,  when  a  fair 
rate  of  increase  was  still  noticeable  which  would  in  the  end  have  carried 
the  figures  up  to  the  next  hundred  in  the  dried  sections.  The  swelling 
of  living  material  in  bog  water  is  but  little  less  than  in  distilled  water 
and  is  also  but  little  different  from  that  in  the  culture  solution,  which 
is  of  the  concentration  used  in  water  cultures.  Hydration  is,  however, 
noticeably  less  in  swamp  water. 

Attention  was  now  turned  to  the  biocolloids  to  ascertain  whether 
the  action  of  plant  material  living  and  dried  would  find  a  parallel  in 
the  action  of  mixtures  of  known  composition.  Sections  of  plates 
composed  of  agar  (90)  and  oat  protein  (10)  were  found  to  show  the 
following  swellings  at  15°C. 

TABLE  60. 

p.  ct. 

Distilled  water 2, 188 

Bog  water 2,083 

Swamp  water 1,200 


Certain  Reactions  of  Biocolloids  and  Cell-masses.  71 

The  decreased  swelling  in  swamp  water  and  the  high  swelling  in  bog 
water  were  marked  and  invariably  shown. 

However,  the  biocolloid  approaches  more  nearly  to  the  condition  of 
the  protoplast  when,  in  common  with  all  living  matter,  it  includes  some 
salts.  The  above  mixture  containing  culture  salts  was  not  avail- 
able, but  some  plates  in  which  the  oat  protein  was  replaced  by  bean 
protein  to  which  had  been  added  0.8  per  cent  of  culture  salts  were 
swelled  in  a  parallel  series.  The  untreated  mixture  free  from  the 
added  salts  does  not  show  as  high  an  imbibition  capacity  as  that  made 
up  with  the  oat  protein.  The  measurements  of  the  increase  of  the 
agar-bean  protein-salted  sections  at  15°  C.  were  as  shown  herewith: 

TABLE  61. 

p.  ct. 

Distilled  water 1 , 525 

Bog  water 1 , 525 

Swamp  water 1 , 100 

Calcium  nitrate,  0.008  M 825 

These  measurements  were  taken  at  the  end  of  40  hours,  while  some 
increase  was  still  in  progress,  but  the  final  relations  would  not  have 
been  materially  altered  by  the  use  of  the  end-points  for  the  compari- 
sons. The  retarding  action  of  the  swamp  water  and  the  equivalence 
of  swelling  in  pure  water  and  bog  water  therefore  runs  plainly  defined 
through  all  of  the  experiments  with  living  and  dried  sections  of  plants 
and  in  tests  with  salted  and  unsalted  biocolloids.  Calcium  nitrate  in 
the  concentration  used  exercises  a  more  marked  effect  on  the  salted 
biocolloid  than  on  the  unsalted  mixture  and  on  the  plant  material. 
The  calcium  content  of  the  biocolloid  is  probably  much  higher  than 
that  of  the  plant,  so  that  the  two  sets  of  measurements  are  not  strictly 
comparable. 

The  calcium  solution  contained  about  eight  times  as  much  salt  as 
the  bog  water  and  nearly  three  times  as  much  as  the  swamp  water, 
which  also  includes  a  trace  of  sodium  chloride.  A  series  was  therefore 
arranged  to  compare  the  action  of  the  two  with  equivalent  salt  solu- 
tions. Sections  of  agar  and  bean  protein  impregnated  with  0.8  per 
cent  of  culture  salts  were  swelled  at  15°  C.  with  results  as  follows: 

TABLE  62. 

p.  ct. 

Distilled  water 1,417 

Bog  water 1 , 444 

Calcium  sulphate  0.048  gram  per  liter 1,417 

Swamp  water 944 

Calcium  sulphate  0.128  gram  per  liter 1 , 083 

Bog  water  and  an  equivalent  calcium  solution  allow  equal  swelling, 
but  the  increase  in  swamp  water  is  much  less  and  also  less  than  in  the 
equivalent  calcium  solution,  to  which  may  be  attributed  most  of  the 
retarding  effect  of  the  swamp  water. 


72  Hydration  and  Growth. 

The  imbibition  capacity  of  the  biocolloids  varies  with  the  propor- 
tions between  the  carbohydrate  and  the  proteins  and  protein  deriva- 
tives. A  biocolloid  was  subsequently  made  up  which  included  a  high 
nitrogen-content  and  a  second  carbohydrate  and  five  albuminous  com- 
pounds. For  this  purpose,  70  parts  agar,  5  parts  each  of  dextrose, 
peptone,  gelatine,  asparagin,  nucleinic  acid,  and  bean  protein  were 
suitably  liquefied  and  poured  into  plates  which  dried  down  to  a 
thickness  of  0.2  mm.  Swellings  of  sections  of  these  plates  at  15°  C. 
gave  the  following  increases  at  the  end  of  24  hours : 

TABLE  63.  p.  ct. 

Distilled  water 725 

Bog  water 592 

Swamp  water 550 

Calcium  chloride,  0.2  M 350 

Calcium  chloride,  0.1  M 400 

The  total  swelling  in  distilled  water  for  this  biocolloid  is  low,  al- 
though it  is  to  be  noted  that  swellings  as  high  as  1,200  per  cent  in  dis- 
tilled water  have  been  measured  at  temperatures  of  18°  to  20°  C. 

Sections  from  plates  made  up  as  above,  but  to  which  had  been 
added  0.8  per  cent  of  culture  salts,  gave  increases  as  follows  at  tem- 
peratures of  15°  C. : 

TABLE  64.  p.  ct. 

Distilled  water. 600 

Bog  water 525 

Swamp  water 625 

This  complex  biocolloid,  high  in  nitrogen  and  in  the  culture  salts, 
displays  hydration  capacity  in  swamp  water  superior  to  that  shown  in 
bog  water  or  pure  water.  The  properties  in  question  would  enable  a 
plant  so  equipped  to  thrive  in  the  waters  of  swamps,  and  it  would  be 
interesting  to  determine  whether  such  a  condition  actually  prevails 
in  the  plants  of  the  sedgy  swamps.1 

The  earlier  attempts  to  interpret  the  swelling  action  of  protoplasm 
were  founded  on  the  assumption  that  such  increase  might  be  repre- 
sented by  the  action  of  gelatine.  The  unsoundness  of  this  assumption 
and  the  inadequacy  of  the  methods  using  this  material  have  been 
amply  demonstrated  by  results  previously  published.  At  one  end  of 
the  scale  stand  some  plants  and  some  plant  structures  high  in  protein- 
aceous  compounds  and  low  in  pentosans,  and  these  do  show  a  behavior 
approximating  that  of  gelatine.  This  is  illustrated  by  the  following 
series,  in  which  sections  of  gelatine  plates  0.18  mm.  in  thickness  were 
swelled  at  15°  C.,  giving  measurements  as  follows: 

TABLE  65.  p.  ct. 

Distilled  water 778 

Bog  water 889 

Swamp  water 939 

Culture  solution,  0.5  per  cent 889 

Potassium  nitrate,  0.01  M 911 

1  Schimper,  A.  F.  W.     Die  Indo-Malayscher  Strandflora,  p.  142.     1891. 


Certain  Reactions  of  Biocolloids  and  Cell-masses.  73 

The  swelling  of  sections  of  agar  plates  0.2  mm.  in  thickness  at  15°  C. 
resulted  in  increases  of: 

TABLE  66. 

p.  ct. 

Distilled  water 700 

Bog  water 650 

Swamp  water 425 

Nutrient  solution,  0.5  per  cent 375 

It  is  to  be  seen  that  all  of  the  solutions  decrease  the  swelling  capacity 
of  the  agar  below  that  displayed  in  distilled  water,  and  that  the  greater 
reduction  in  the  nutrient  solution  is  to  be  attributed  to  the  higher  salt- 
content. 

Plants  of  bogs  and  especially  swamps  are  undoubtedly  subjected  to 
great  variations  in  the  composition  of  the  water  by  reason  of  inunda- 
tions and  floods.  It  was  thought  pertinent  to  extend  experiments 
in  which  alternations  of  solutions  were  made  in  such  manner 
as  to  test  the  effect  of  previous  history  on  the  behavior  of  a  bio- 
colloid.  Sections  of  agar-oat  protein  0.18  mm.  in  thickness  swelled 
972  per  cent  in  12  hours  at  17°  to  19°  C.  and  reached  a  total  of  1,233 
per  cent  at  the  end-point  in  108  hours,  which  are  equivalent  to  results 
previously  attained  and  hence  afford  a  fair  basis  of  comparison  with 
the  following,  in  which  a  trio  of  sections  swelled  2,361  per  cent  in  dis- 
tilled water  at  the  end-point  in  72  hours.  Replacement  of  distilled 
water  with  swamp  water  was  followed  by  a  slow  shrinkage,  but  this 
amounted  to  only  36  per  cent  of  the  original  volume.  No  swelling 
agent  yet  tested  has  been  found  to  reverse  the  action  of  another  solu- 
tion so  fully  as  to  bring  the  dimensions  of  the  sections  down  to  the 
dimensions  which  might  be  attained  in  the  second  agent  alone. 

Sections  of  a  biocolloid  consisting  of  90  parts  agar  and  10  parts  of 
bean  protein  to  which  0.8  per  cent  of  nutrient  salts  had  been  added, 
0.18  mm.  in  thickness,  were  now  swelled  in  swamp  water  at  18°  to 
20°  C.  The  increase  was  measured  at  the  end  of  16  hours,  at  which 
time  the  total  swelling  was  1,082  per  cent.  The  swamp  water  was 
now  replaced  with  hundredth-normal  citric  acid-potassium  nitrate  for 
36  hours,  during  which  time  no  appreciable  change  was  registered. 
Replacement  of  this  solution  with  swamp  water  was  followed  by  a 
resumption  of  the  swelling,  which  carried  the  thickness  of  the  sections 
to  1,388  per  cent  of  the  original,  which  is  greater  than  that  attained 
in  the  simple  continuous  swelling  in  swamp  water. 

Swamp  water  is  high  in  salts,  and  it  is  probably  this  feature  to  which 
its  influence  on  swelling  is  due.  A  test  parallel  to  the  above  was  made 
in  which  the  sections  were  first  swelled  in  a  0.5  per  cent  nutrient  solu- 
tion in  which  the  salts  are  somewhat  more  concentrated  than  in  the. 
swamp  water.  A  swelling  of  888  per  cent  took  place  in  17  hours,  at 
which  time  the  pen  of  the  auxograph  was  tracing  a  horizontal  line. 
Replacement  of  the  nutrient  solution  with  the  acidified  potassium- 


74  Hydration  and  Growth. 

nitrate  solution  was  followed  by  a  very  slight  swelling.  When  this 
solution  was  replaced  by  swamp  water  an  increase  of  333  per  cent 
followed  in  40  hours.  The  two  tests  were  parallel,  except  that  the 
initial  treatment  in  one  case  was  with  nutrient  solution  in  which  the 
salts  were  more  concentrated  than  in  the  swamp  water  used  in  the 
other.  The  final  swelling  in  the  second  case  is  greater  and  may  be 
attributed  to  the  initial  salt  action. 

In  a  partial  repetition  of  the  above  test,  the  sections  placed  in  culture 
solution  swelled  861  per  cent  in  20  hours.  Lengthening  the  immersion 
in  acidified  potassium  nitrate  to  55  hours  was  accompanied  by  a 
swelling  of  55  per  cent.  Replacement  with  distilled  water  set  up  a  slow 
increase  which  resulted  in  a  gain  of  111  per  cent  in  thickness  in  40 
hours.  The  total  increase  was  1,027  per  cent,  while  the  one  finished 
in  swamp  water  swelled  1,388  per  cent. 

The  relative  effects  of  swamp  and  bog  water  on  biocolloids  were 
tested  in  still  another  way.  Plates  of  agar-oat  protein  were  prepared 
in  which  strips  of  webbing  of  cotton  were  embedded  in  the  soft  material 
as  it  cooled  for  the  purpose  of  testing  the  influence  of  certain  purely 
mechanical  features  on  swelling.  Portions  of  the  plates  dried  down  to 
a  thickness  of  0.18  mm.  in  the  clear  portion  of  the  plate  and  sections 
from  this  swelled  2,111  per  cent  in  bog  water  and  1,195  per  cent  in 
swamp  water  at  15°  C.  Sections  containing  webbing  were  0.58  mm. 
in  thickness  and  the  actual  increase  of  such  sections  was  491  per  cent 
in  bog  water  and  distilled  water  and  371  per  cent  in  swamp  water. 
If  the  increase  be  calculated  on  the  assumption  that  the  webbed  sec- 
tions included  as  much  biocolloid  as  the  free  sections,  the  proportions 
would  be  1,583  per  cent  in  bog  water  and  distilled  water  and  1,195 
per  cent  in  swamp  water. 

Swamp  water  has  been  found  to  affect  absorption  and  swelling  in  the 
same  manner  as  an  equivalent  solution  of  calcium  sulphate.  Swelling 
and  absorption  is  retarded  by  swamp  water  in  salted  biocolloids  and 
in  sections  of  plants  containing  a  large  proportion  of  pentosans  and  a 
low  protein-content.  Biocolloids  with  a  high  protein  and  salt  content, 
on  the  other  hand,  show  an  enhanced  absorption  in  swamp  water. 
Inferentially,  plants  of  similar  constitution  would  carry  on  absorption 
readily  and  thrive  in  swamp  waters..  Whether  adaptation  to  swamp 
habitats  actually  takes  this  course  is  not  known. 

An  extension  of  these  measurements  was  made  to  ascertain  the 
effects  of  water  and  soil  solutions  which  were  in  common  use  at  the 
Desert  Laboratory  upon  a  biocolloid,  a  mixture  consisting  of  6  parts 
of  agar,  2  parts  of  mucilage  of  Opuntia,  1  part  of  gelatine,  and  1  part 
of  bean  protein.  This  had  been  poured  in  the  usual  manner  and  dried 
to  a  thickness  of  0.2  mm.  Sections  of  the  usual  size  were  placed  in 
dishes  under  the  auxograph.  Distilled  water  of  the  grade  used  in 
making  up  all  of  the  solutions  caused  a  swelling  of  1,750  per  cent;  ram 


Certain  Reactions  of  Biocolloids  and  Cell-masses.  75 

water  which  had  fallen  on  a  slate  roof  and  collected  in  a  closed  cement 
cistern  gave  a  swelling  of  1,500  per  cent.  The  water  from  the  system 
supplying  the  Desert  Laboratory,  taken  from  a  well  40  feet  in  depth 
in  the  alluvium  of  the  flats  along  the  Santa  Cruz  River  and  pumped 
through  an  iron  pipe  line  6,000  feet  long  to  a  cement  tank,  produced  a 
swelling  of  only  800  per  cent.  As  a  final  test,  a  soil  solution  was  used 
which  was  obtained  by  shaking  up  600  grams  of  surface  soil  with  1,200 
c.c.  of  distilled  water  and  then  allowed  to  stand  for  12  hours.  The 
filtered  solution  applied  to  sections  in  the  same  manner  as  the  other 
waters  induced  a  hydration  of  900  per  cent.  (Fig.  10). 

These  measurements  afford  a  standard  of  desirability  of  the  water 
from  these  various  sources  for  cultural  work  and  for  drinking  purposes. 
Since  growth  consists  in  the  main  of  the  hydration  of  plasmatic  col- 
loids, the  nutritive  solution  most  favorable  to  this  process  would  be  an 
important  factor  in  an  environmental  optimum.  It  was  also  possible 
to  make  tests  of  these  natural  waters  with  a  biocolloid  which  included 
6  parts  agar,  2  parts  prosopis  gum,  1  part  gelatine,  and  1  part  bean 
protein,  to  which  had  been  added  0.2  per  cent  of  culture  solution. 
Such  a  mixture,  like  one  containing  gum  arabic,  shows  high  swelling 
in  acids  and  less  in  salts,  whether  acid  or  alkaline.  Swelling  of  plates 
0.17  mm.  in  thickness  were  as  shown  herewith: 

TABLE  67. 

p.  ct. 

Distilled  water  (25°  C.) 1,760 

Rain  water  (27°  C.) 1,794 

Well  water  (27°  C.) 1,706 

Soil  water  (25°  C.) 1,500 

The  higher  temperatures  at  which  the  swellings  in  rain  water  and 
well  water  were  made  prevents  direct  comparison,  but  it  may  be  sup- 
posed that  a  biocolloid  already  charged  with  salts  to  a  point  above  the 
average  of  land  plants  would  be  hydrated  in  the  dilute  solution 
offered  by  the  cistern  water  practically  as  readily  as  from  distilled 
water.  The  figure  given  expresses  the  increase  at  a  temperature  2°  C. 
higher.  The  same  would  be  true  of  the  well  water  as  compared  with 
the  soil  solution.  It  is  to  be  noted  that  the  difference  between  the 
reaction  in  the  solutions  and  in  pure  water  is  less  than  in  the  unsalted 
colloid.  Of  course,  the  substitution  of  prosopis  gum  for  opuntia 
mucilage  is  also  a  factor.  Relations  to  environmental  conditions  of 
some  importance  are  suggested.  The  reactions  of  the  halophytes 
should  include  some  effects  similar,  in  that  there  would  be  offered 
the  phenomena  of  the  swelling  of  cell-masses  high  in  salts  (fig.  10). 

Some  experiments  in  the  modification  of  germ-plasm  in  1905  resulted 
in  the  formation  of  embryos  developing,  into  individuals  not  entirely 
identical  with  the  parental  types.  The  essential  feature  of  the  experi- 
ment consisted  in  the  successful  introduction  of  various  substances 
into  the  neighborhood  of  the  embryo-sacs  at  the  time  that  fertilization 


76 


Hydration  and  Growth. 


was  imminent,  and  when  the  first  trials  were  made  I  had  two  main 
purposes  in  mind :  first,  to  ascertain  whether  or  not  foreign  substances 
could  be  introduced  into  ovaries  in  such  manner  as  to  affect  the  ovules 
with  a  minimum  of  traumatic  effects,  so  that  the  ovaries  might  reach 
maturity;  and  secondly,  to  ascertain  whether  or  not  such  changes 
could  be  produced  in  an  early  stage  of  sexual  specialization  before  the 
development  of  the  embryo-sac  or  after  the  union  of  the  sexual  ele- 
ments in  fertilization.  The  value  of  the  results  was  much  lessened  by 
the  fact  that  the  direct  effects  of  the  reagents  could  not  be  identified. 
After  some  difficulty  the  actual  diffusion  of  the  liquids  in  the  ovaries 
was  ascertained  by  substituting  dyes  for  the  salt  solutions,  but  there 
still  remains  to  be  determined  the  nature  of  the  action  of  the  reagents 
on  the  cell  colloids. 


12  p.m. 


FIG.  10. — Tracing  of  auxographic  record  of  swelling  of  plates  consisting  of  6  parts  agar,  2  parts 
mucilage  of  Opuntia,  1  of  gelatine,  and  1  of  bean  protein,  in  distilled  water,  A ;  rain  water,  B; 
well  water,  C;  and  a  soil  solution,  D. 

The  first  step  hi  such  an  examination  would  naturally  be  the  measure- 
ment of  the  hydration  effect.  Plates  of  agar  90  parts  and  bean  pro- 
tein 10  parts,  0.25  mm.  thick,  were  swelled  to  ascertain  the  possible 
imbibition  effects.  A  series  in  the  dark  room  at  15°  to  16°  C.  gave  the 
following  measurements  of  expansion : 

TABLE  68. 

p.  ct. 

DistUled  water 1,220 

Methylene  blue 860 

Iodine,  sat.  solution  in  distilled  water 1 , 080 

A  third  reagent  used  in  the  later  series  of  ovarial  treatments,  zinc 
sulphate,  was  tested  in  the  concentration  of  1  part  in  10,000  (0.00034  M) 
in  comparison  with  distilled  water  and  hydroxid  on  sections  of  agar- 
bean  albumin.  The  swellings  were  as  below : 

TABLE  69. 

p.  ct. 

Distilled  water 1,388 

Zinc  sulphate,  0.00034  M 833 

Sodium  hydroxid,  0.01  M 194 

The  characteristic  high  rate  of  swelling  of  agar-albumin  is  exhibited, 
the  swelling  in  the  hydroxid  being  but  one-seventh  that  in  water.  The 


Certain  Reactions  of  Biocolloids  and  Cell-masses.  77 

zinc  salt,  although  very  dilute,  retards  swelling  noticeably,  and  exerts 
a  greater  effect  on  the  imbibition  capacity  than  does  either  of  the 
other  reagents  named.1 

Whatever  value  may  attach  to  this  procedure,  it  seems  reasonable 
to  assume  that  salts  less  toxic  in  effect  than  those  of  zinc  and  identical 
with  those  already  present  in  the  embryo-sac  offer  the  greatest  promise, 
and  their  most  intense  effect  might  be  secured  when  acidified. 

The  presence  of  free  amino-groups  in  the  cell  and  their  rapid  penetra- 
tion of  plasmatic  structures  makes  it  highly  probable  that  some  of 
these  substances  singly  or  in  combination  might  diffuse  throughout  the 
cytoplasmic  structure  of  the  embryo-sac  and  also  reach  the  chromo- 
somes with  a  high  degree  of  possibility  of  affecting  then*  genetic  con- 
tent or  potentiality. 

1  MacDougal  and  Spoehr.  The  effect  of  organic  acids  and  their  amino-compounds  on  the  hydra- 
tion  of  agar  and  on  a  biocolloid.  Proc.  Soc.  Exper.  Biol.  and  Med.,  16:33.  1918. 

MacDougal,  D.  T.  The  experimental  modification  of  germ-plasm.  Annals  Mo.  Bot.  Garden, 
2:253-274,  Feb.-Apr.,  1915. 


VII.  FLUCTUATING  OR  ALTERNATING  HYDRATION  EFFECTS. 
BASIS  OF  XEROPHILY  AND  SUCCULENCE. 

The  experiments  described  in  the  previous  chapters  have  dealt 
chiefly  with  sections  of  colloidal  material  artificially  compounded  to 
represent  the  materials  and  conditions  which  affect  hydration  in  plants. 
Measurements  of  the  swelling  of  dried  sections  of  this  material  have 
been  used  as  a  basis  for  comparison  with  the  action  of  slices  or  sections 
of  plant  cell-masses  similarly  dehydrated  or  in  living  condition. 

No  estimate  of  results  of  this  kind  will  be  valid  and  no  perspective 
of  their  bearing  will  be  correct  which  does  not  take  into  account 
the  fact  that  the  growing  parts  of  the  higher  plants  contain  90  per 
cent  or  more  water  and  that  the  colloids  of  the  protoplast,  the  action 
of  which  makes  for  the  distension  or  enlargement  of  growth,  are  even 
more  highly  hydrated.  These  gels  also  invariably  contain  the  culture 
salts,  in  combination  or  simply  adsorbed,  and  are  inevitably  in  a  con- 
dition of  acidity  resulting  from  their  carbohydrate  metabolism. 

The  elementary  facts  obtained  by  the  experiments  described  in  the 
foregoing  pages  made  it  possible  to  plan  a  series  of  treatments  of 
hydrating  material  in  which  the  previous  experience  of  biocolloids 
would  be  apprehended  in  swellings  in  salts,  acids,  etc.,  in  a  sequence  of 
interest  to  the  physiologist,  and  to  obtain  additive,  alternating,  or 
superposed  effects.  Variations  in  the  carbohydrate  and  proteinaceous 
substances  of  a  colloidal  mixture  may  not  be  readily  produced  to 
simulate  the  changes  which  form  the  basis  of  some  of  the  most  funda- 
mentally important  phenomena  of  the  cell.  The  worker  must  approach 
this  phase  of  the  subject  by  studying  the  reactions  of  separate  masses 
or  lots  of  material  compounded  to  represent  various  stages  in  the 
condition  of  the  protoplasm.  Something  of  this  kind  has  been  done 
in  the  measurement  of  the  reactions  of  biocolloids  of  several  kinds.  It 
is  of  course  impossible  to  imitate  any  of  the  important  metabolic 
processes  which  make  cell-colloids  continuously  acting  machines,  al- 
though no  hint  has  been  found  of  any  source  of  energy  or  directive 
action  outside  of  surface  tension  and  chemical  action. 

In  the  subjection  of  colloidal  masses  to  the  action  of  hydrating 
solutions,  as  described  in  the  following  pages,  it  is  obvious  that  the 
soluble  constituents  of  the  sections  would  be  partially  and  unequally 
removed  with  every  change  in  the  solutions  in  which  they  were  sub- 
merged, and  while  the  fact  that  the  colloidal  mass  changed  its  com- 
position continuously  during  the  immersion  in  the  various  reagents, 
yet  it  can  not  be  said  that  these  alterations  were  identical  with  those 
of  the  growing  cell.1  Some  highly  suggestive  results  or  situations  were 
produced,  however,  by  the  replacements  of  hydrating  solutions,  as 
described  in  following  pages. 

NOTE. — All  measurements  of  swelling  and  shrinkage  are  given  in  terms  of  original  or  dried 
thickness  of  sections. — AUTHOR. 

1  MacDougal  and  Spoehr.     The  effects  of  acids  and  salts  on  biocolloids.     Science,  46 : 269.     1917. 
78 


Fluctuating  or  Alternating  Hydration  Effects. 


79 


An  experiment  of  this  kind  was  carried  out  July  30  to  August  9, 
1917,  in  the  equable-temperature  chambers  of  the  Coastal  Laboratory 
at  15°  to  16°  C.,  in  which  sections  composed  of  9  parts  agar  and  1  part 
bean  protein  were  subjected  to  alternating  action  of  acids  and  hydro- 
xid  after  they  had  first  been  swelled  in  water  for  5  hours.  Citric,  malic, 
and  formic  acids  were  used  in  separate  sets  at  hundredth-normal  con- 
centration, but  no  determination  was  made  of  the  hydrogen-ion  con- 
centration, and  as  the  initial  swellings  in  water  were  widely  divergent, 
the  final  totals  have  no  especial  significance,  entire  interest  lying  in  the 
changes  in  volume  resulting  from  the  replacements.  (See  fig.  11.) 


I2pm.    m.      12p.m.    m.      12p.m.    m.     12p.m.     m.     12p.m.     m.     12p.m.     m.      12p.m. 
~r 7 f r r 1 r ? 7 r T /         '• 


FIG.  11. —  Record  of  variations  in  thickness  of  sections  of  agar  and  bean  protein  subjected  to  the 
action  of  water,  acids,  and  alkalies,  as  described  in  text  pages  79  and  80. 

Trios  of  sections  which  were  0.25  mm.  in  thickness  were  placed  in 
dishes  into  which  distilled  water  was  poured  in  the  usual  manner. 
At  the  end  of  5  hours,  designated  as  A  on  the  tracing,  the  water  was 
drawn  off  by  a  pipette  and  a  solution  of  hundredth-normal  sodium 
hydroxid  substituted,  which  was  followed  by  an  expansion  which 
reached  almost  to  the  possible  total  of  580  to  860  per  cent  of  the 
original  dried  sections  in  8  hours.  At  the  end  of  this  time  the  sections 
were  in  an  advanced  stage  of  hydration  and  were  also  probably 
impregnated  with  salts  formed  by  potassium  with  the  carbohydrates 
and  proteins  in  the  gel,  and  the  possible  presence  of  these  compounds 
as  modified  by  subsequent  experiences  must  be  kept  in  mind. 

The  three  acids  were  now  added  to  the  separate  sets  of  sections, 
and  from  this  point  their  experiences  diverge.  All  agreed  in  under- 
going a  retraction  during  the  next  9  hours  which  was  most  pro- 
nounced in  the  citric  acid.  Shrinkage  at  a  slow  rate  was  still  in  prog- 
ress at  the  end  of  this  period,  B,  when  the  acid  solutions  were  removed 
and  distilled  water  substituted.  No  effort  was  made  to  wash  the 
sections  which  were  saturated  with  the  acid  and  salt,  and  the  operation 
resulted  simply  in  a  reduced  acidity  in  which  some  slight  swelling  took 
place  during  the  5  hours  ending  at  C,  at  which  time  the  reaction  was 
practically  at  an  end. 

The  hydroxid  was  now  used  for  the  second  time,  replacing  the  acid, 
for  2  hours,  in  which  time  a  further  slight  swelling  ensued.  Acids 


80  Hydration  and  Growth. 

were  again  used  at  D  and  a  slow  shrinkage  again  set  in  which  fol- 
lowed on  during  the  18  hours  until  the  change  was  made  at  E  to 
hydroxid,  which,  as  before,  simply  neutralized  the  acid  and  increased 
the  salt-content,  with  the  result  that  the  volume  of  the  colloid  swelled 
to  the  dimensions  occupied  before  the  last  treatment  with  acid.  At 
the  end  of  the  5  hours,  F,  the  hydroxid  was  removed  and  water,  re- 
newed once,  poured  into  the  dishes.  The  effect  was  very  marked, 
as  a  rapid  swelling  ensued  during  the  next  4  hours,  at  the  end 
of  which  time  it  was  in  progress  at  an  undiminished  rate,  being  greatest 
in  the  formic  and  least  in  the  citric  acid.  The  sections  now  contained 
a  large  proportion  of  water,  sufficient  to  bring  them  into  the  condition 
of  hydration  of  active  protoplasm,  and  the  addition  of  the  acids  at  G 
arrested  the  swelling  abruptly  and  caused  a  shrinkage  which  was  di- 
minishing at  the  end  of  4  hours.  The  shrinkage  was  checked  by  replace- 
ment with  water  at  H  and  a  slight  swelling  took  place  in  the  ensuing 
11  hours. 

The  water  now  being  replaced  by  hydroxid,  a  sudden  slight  ex- 
pansion occurred  in  all  of  the  sections,  followed  by  a  shrinkage  which 
had  not  ceased  entirely  at  the  end  of  9  hours,  during  which  period 
additional  salts  were  being  formed  in  the  colloidal  structure.  Now, 
when  the  hydroxid  was  partially  washed  out  by  water  at  J,  a  swelling 
ensued  which  in  15  hours  brought  the  sections  very  nearly  to  their 
final  thickness  and  consequently  near  their  maximum  imbibition 
capacity.  It  is  in  this  condition,  of  course,  that  growing  protoplasts 
are  normally  active.  The  greatest  expansion  in  this  phase  was  in  the 
material  treated  previously  with  malic  acid. 

The  addition  of  hydroxid  (K)  was  now  followed  by  a  marked 
shrinkage  which  had  not  ceased  at  the  end  of  8  hours.  Replace- 
ment of  the  hydroxid  with  acid  at  L  caused  a  further  abrupt 
retraction  which  soon  ceased,  and  after  5  hours  the  acid  was  re- 
placed with  water  once  renewed  (M),  only  a  slight  swelling  resulting 
during  the  next  10  hours.  Again  hydroxid  (N)  caused  a  sudden 
expansion  to  be  followed  by  a  slow  shrinkage  which  had  not  reached 
its  end  in  13  hours.  Water  now  following  hydroxid  at  0,  a  much 
greater  expansion  took  place  than  when  water  followed  acid.  Alka- 
losis  at  P  now  came  after  an  hydroxid-water  period  and  was  not 
followed  by  the  abrupt  enlargement  consequent  upon  adding  hy- 
droxid to  the  colloid  after  an  acid-water  period.  The  shrinkage, 
however,  was  as  marked  as  in  previous  experiences,  and  had  not  ended 
in  8  hours. 

Replacement  with  water  at  S  was  followed  by  an  expansion  which 
had  not  ceased  at  the  end  of  6  hours.  Finally,  acids  caused  a  diminu- 
tion the  most  marked  of  any  produced  by  these  substances.  Both 
acids  and  hydroxids  caused  the  most  marked  changes  ni  the  highly 
hydrated  and  salted  sections  near  the  end  of  the  week  over  which  the 


Fluctuating  or  Alternating  Hydration  Effects. 


81 


experiments  had  been  extended.  It  is  to  be  noted  that,  in  addition  to 
hydration  and  possible  salt  formation,  the  colloid  was  also  undergoing 
some  alteration  by  the  unequal  solution  out  of  the  solution  of  pro- 
tein and  agar. 


Fio.  12. 

Continuation  of  record 
of  variations  of  sec- 
tions of  agar  and  bean 
protein  in  fig.  11.  For 
description  see  text, 
pages  81  and  82. 


12p.m.         m.          I2p.m 


12p.m. 


12p.m. 


New  sheets  were  fitted  to  the  recorder  of  the  auxograph  and  arrange- 
ments made  to  follow  further  changes  (see  fig.  12).  During  the  next 
4  days  an  additional  swelling  of  280  per  cent  in  the  malic  series,  320 
per  cent  in  the  citric,  and  300  per  cent  in  the  formic  were  recorded. 
Replacement  of  the  acid  by  hydroxid  (1)  resulted  first  in  an  expan- 
sion which  was  partially  lost  in  7  hours,  so  that  the  net  gain  was  very- 
light.  When  the  hydroxid  was  washed  off  (2)  hydration  in  distilled 
water  was  followed  by  expansion  of  lesser  amplitude  than  in  the 
previous  procedure  of  this  kind,  but  it  had  not  ceased  at  the  end 
of  14  hours.  A  diminution  in  each  repetition  was  found.  Hydroxid 
(3)  failed  to  bring  the  sections  back  to  the  dimensions  preceding  the 
last  hydration.  Replacement  of  the  hydroxid  by  acid  (4)  caused  a 
further  slight  contraction,  but  not  to  the  last  pre-hydration  dimen- 
sions. In  fact,  every  hydration  included  an  irreversible  element. 
Hydroxid  (5)  again  produced  shrinkage,  and  then  contraction  which 
soon  ceased.  After  13  hours  in  hydroxid,  water  applied  and  re- 
newed (6)  produced  a  swelling  which  was  in  progress  at  the  end  of 
12  hours.  Replacement  with  acids  (7)  was  followed  by  very  abrupt 
shrinkages  which  were  more  gradual  in  formic  acid.  Substitution 
of  hydroxid  after  11  hours  at  8  was  followed  by  the  expected  initial 
expansion  and  subsequent  shrinkage.  The  final  hydration  (9)  on  the 
tenth  day  of  the  test  gave  swellings  with  net  expansions  of  7,  9,  and 
8,  as  compared  with  21,  18,  and  17  on  the  sixth  day.  The  biocolloid 
is  thus  seen  to  progress  through  a  period  of  reactions  of  increasing 
amplitude  to  a  climax,  followed  by  one  of  diminishing  alterations  in 


82 


Hydration  and  Growth. 


water.  Such  progress  is  probably  accompanied  by,  or  consequent 
upon,  changes  in  the  proteins  and  in  the  pentosans.  On  the  other 
hand,  as  the  sections  come  nearer  to  their  total  imbibitional  or 
hydration  capacity,  they  are  more  sensitive  to  acidity  and  not  only 
the  speed  but  the  amount  of  retraction  increases. 

Another  series  was  based  on  the  behavior  of  an  agar  (90  parts)  bean 
albumen  (10  parts)  mixture  in  plates  0.18  mm.  in  thickness.  Three 
sets  of  sections  were  given  treatment  as  nearly  identical  as  possible, 
a  special  feature  being  made  of  the  action  of  salts. 

An  initial  swelling  of  1,055  to  1,222  per  cent,  averaging  1,137  per 
cent,  was  first  produced  in  7  hours  in  distilled  water,  at  which  tune 
it  is  evident  the  plates  had  increased  to  a  thickness  of  .about  2  mm.,  or 
one-half  of  the  total  displayed  in  the  test.  (See  fig.  13.)  The  replace- 


m.      l?p.m.      m.      12p.m.     m.       I?  p.m 


12p.m. 


12p.m.     m       12  p.  m 


12p.m.      m. 


Fio.  13. — Variations  in  thickness  in  sections  of  agar  and  bean  albumen  subjected   to  the  action 

of  acids,  salts,  and  hydroxid. 

ment  of  the  water  by  a  hundredth-molar  solution  of  potassium  nitrate 
(A)  for  14  hours  was  characterized  by  a  slight  initial  shrinkage,  followed 
by  very  slow  swelling,  which  was  probably  accompanied  by  a  solution 
of  the  albumin  from  the  sections.  Replacement  of  the  salt  solution 
.by  another  one  acidified  by  a  hundredth-normal  citric-acid  solution  (B), 
produced  a  shrinkage  which  brought  the  dimensions  of  the  sections 
to  about  that  at  the  end  of  the  initial  swelling  in  water.  The  material 
might  now  be  considered  as  a  biocolloid  containing  some  salt  and 
acidified  to  a  point  probably  equivalent  to  conditions  in  living  material. 
Replacement  of  the  acidified  salt  with  alkaline  salt  of  the  same  con- 
centration resulted  in  a  swelling  averaging  200  per  cent  of  the  original 
thickness  of  the  dried  plate. 

The  sections  were  now  washed  (D)  and  allowed  to  swell  for  9  hours  in 
distilled  water,  making  an  increase  of  over  1,100  per  cent  and,  as  will 
be  obvious,  bringing  the  water-content  of  the  biocolloid  to  a  point 
nearing  the  maximum  capacity. 

Replacement  of  water  by  an  alkaline  salt  solution  (E)  now  pro- 
duced a  greater  shrinkage  than  when  the  biocolloid  had  a  water-con- 
tent about  a  half  less,  the  loss  being  200  per  cent  of  the  original  thick- 


Fluctuating  or  Alternating  Hydration  Effects.  83 

ness.  Replacement  with  acid  (F)  resulted  in  a  further  slight  shrink- 
age, no  noticeable  loss  ensuing  when  the  acid  was  replaced  by  acidified 
salt  (<?).  Replacement  with  alkaline  salt  (H)  was  followed  by  an  in- 
crease which  had  ceased  at  the  end  of  4  hours.  Water  now  caused  a 
slow  continued  swelling  (/),  at  which  time  the  maximum  size  of  the 
sections  was  reached  and  they  had  swelled  2,400  to  2,800  per  cent  of 
the  diameter  of  the  dried  plate. 

Replacement  of  the  water  by  an  acidified  solution  (J)  resulted  in  a 
very  rapid  shrinkage,  which  was  nearly  complete  in  an  hour  and  re- 
duced the  sections  nearly  200  per  cent  of  the  original.  Replacement 
of  the  acidified  salt  by  water  (K),  which  was  renewed  three  tunes, 
regained  only  about  half  of  the  thickness  lost  in  the  acidified  salt.  It 
is  to  be  remembered  that  the  sections  were  in  a  condition  of  acidosis, 
and  this  would  not  be  reduced  below  a  certain  point  until  alkali  was 
introduced.  This  was  done  in  an  alkaline  salt  (L),  which  produced  a 
very  slight  shrinkage.  The  swelling  in  water  following  this  treatment 
was  of  such  reduced  amplitude  that  the  measurements  were  discon- 
tinued. The  successive  treatments  had  resulted  in  the  incorporation 
of  water  to  the  full  imbibition  capacity  of  the  colloid,  and  the  reagents 
had  produced  such  changes  that  mere  variations  in  acidity,  alkalinity, 
etc.,  caused  but  little  variation  in  this  amount  and  in  the  volume  of 
the  sections,  a  condition  in  general  analogous  to  that  of  a  mature  organ. 

A  series  of  tests  were  now  planned  to  induce  alterations  in  sections 
in  which  imbibition  was  first  carried  to  a  degree  near  the  saturation- 
point.  Sections  of  agar  90  parts,  bean  protein  10  parts,  0.16  mm.  in 
thickness,  swelled  2,156  per  cent  in  18.5  hours  at  16°  to  18°  C.,  and 
the  rate  of  expansion  had  fallen  very  low.  Substitution  of  a  hun- 
dredth-normal potassium-nitrate  solution  checked  further  increase  and 
induced  a  very  slight  shrinkage  in  6.5  hours,  and  no  perceptible  alter- 
ation took  place  by  the  substitution  of  citric  acid  (0.01  M)  for  15.5 
hours.  Replacement  with  potassium  hydroxid  +  potassium  nitrate 
(0.01  M)  resulted  in  a  swelling  of  156  per  cent  of  the  original  dimen- 
sions in  8  hours,  at  the  end  of  which  time  this  action  had  entirely 
terminated.  The  sections  now  being  in  an  alkaline  condition,  their 
immersion  in  water  was  followed  by  an  increase  of  1,250  per  cent  of 
the  original  dimensions,  the  total  now  being  3,406  per  cent. 

Replacement  of  the  water  by  a  hundredth-normal  solution  of  potas- 
sium nitrate  and  citric  acid  resulted  in  a  greater  change  than  at  a 
lower  water-content,  the  shrinkage  now  being  190  per  cent  of  the  origi- 
nal diameter  of  the  air-dry  plate.  When  the  sections  had  swelled  but 
2,156  per  cent,  the  action  of  a  similar  solution  did  but  little  more  than 
check  swelling. 

The  biocolloid  at  this  last  point  was  equivalent  to  a  protoplasm  in 
its  water-relations  which  contained  over  95  per  cent  water  and  less 
than  5  per  cent  dry  matter.  In  a  later  stage  the  biocolloid  was  made 


84  Hydration  and  Growth. 

up  of  less  than  3  per  cent  dry  matter  and  more  than  97  per  cent 
water,  and  hence  approximated  the  dispersion  of  active  protoplasm. 

Other  sections  from  the  same  plate  swelled  2,094  per  cent  in  18.5 
hours  at  16°  to  18°  C.  The  use  of  potassium  nitrate  and  citric  acid 
(0.01  N)  resulted  in  a  shrinkage  of  less  than  a  hundred  per  cent  within 
an  hour,  after  which  the  volume  was  stationary.  As  the  experiences 
of  the  two  sets  of  sections  had  been  identical  hitherto,  it  is  interesting 
to  note  that  in  this  stage  of  imbibition  (95  per  cent  water)  acidified 
salt  produced  more  shrinkage  than  the  salt  alone,  a  result  in  harmony 
with  those  obtained  by  swelling  biocolloids.  Imbibition  in  the  salt 
being  generally  greater  than  in  the  acidified  salt,  replacement  of  the 
acidified  salt  with  the  alkaline  salt  was  followed  by  a  swelling  amount- 
ing to  over  300  per  cent  of  the  original  thickness  of  the  sections  in  9 
hours,  at  the  end  of  which  time  the  swelling  had  not  reached  its  limit. 
Replacement  with  water  speeded  the  imbibition  to  a  rate  which  made 
an  increase  of  over  800  per  cent  in  9  hours.  The  sections  now  con- 
tained nearly  97  per  cent  water,  having  swelled  3,156  per  cent,  and 
when  the  water  in  the  dish  was  replaced  with  acidified  potassium 
nitrate  (0.01  M),  the  shrinkage  was  greater  than  in  the  previous  experi- 
ence, now  amounting  to  300  per  cent  of  the  original  thickness  of  the 
sections.  A  further  shrinkage  followed  the  substitution  of  alkaline 
salt  solution. 

Tests  were  arranged  to  ascertain  the  effect  of  the  initial  hydration 
medium,  using  salts  and  acidified  salts.  Trios  of  sections  of  agar  90 
parts  and  oat  protein  10  parts,  0.18  mm.  in  thickness,  were  put  under 
the  auxograph  in  a  darkened  chamber  which  remained  steadily  at  18° 
C.  during  the  week  in  which  the  measurements  were  made. 

The  sections  which  were  immersed  in  hundredth-molar  potassium 
nitrate  reached  a  size  within  100  per  cent  of  the  possible  total  in 
16.5  hours,  at  which  time  the  expansion  was  about  1,200  per  cent. 
The  replacement  of  the  salt  by  distilled  water  which  was  renewed 
twice  induced  an  additional  expansion  of  1,750  per  cent  in  22.5  hours, 
which  was  the  practical  limit  of  the  sections.  Replacement  with 
hundredth-normal  acidified  potassium  nitrate  resulted  in  a  shrink- 
age of  about  100  per  cent,  the  movement  being  complete  in  6  hours. 
Replacement  of  the  acidified  salt  with  water  (renewed  three  times) 
induced  a  slow,  long-continued  swelling,  which,  at  the  end  of  18.5  hours, 
had  resulted  in  an  increase  of  about  100  per  cent,  and  which  carried 
the  sections  back  to  the  maximum  thickness  attained  before  the  acidi- 
fied salt  was  added.  The  water  was  now  replaced  by  a  simple  hun- 
dredth-normal potassium-nitrate  solution,  which  produced  a  shrinkage 
slightly  less  than  that  displayed  24  hours  before  in  acidified  salt.  This 
was  followed  by  a  long-continued  swelling,  which  again,  in  21  hours, 
brought  the  sections  to  the  maximum  thickness.  The  change  to  an 
acidified  salt  solution  resulted  in  a  shrinkage  about  equivalent  to  the 


Fluctuating  or  Alternating  Hydration  Effects.  85 

previous  one,  and  the  experiment  was  discontinued  after  a  total  period 
of  100  hours. 

The  use  of  hundredth-normal  potassium  nitrate-citric  acid  as  the  first 
solution  caused  a  swelling  of  708  per  cent  in  7  hours,  at  which  time 
the  rate  of  increase  was  very  slow  and  would  not  have  carried  the 
thickness  to  more  than  800  per  cent  of  the  original.  Replacement  of 
the  acidified  salt  by  the  salt  alone  in  the  same  concentration  and  its 
renewal  accelerated  imbibition,  which  proceeded  at  a  rate  which  les- 
sened very  slowly  for  63  hours.  The  swelling  was  at  first  at  the  rate 
of  100  per  cent  in  4  hours  and  about  25  per  cent  during  the  last  4  hours. 
The  increase  during  this  period  of  87  hours  was  about  700  per  cent, 
making  the  total  increase  over  1,400  per  cent.  The  behavior  of  the 
colloid  during  this  period  was  that  of  a  plant  with  diminishing  acidity. 

The  salt  was  now  pipetted  off  and  replaced  with  distilled  water  which 
was  renewed.  The  acceleration  following  was  so  abrupt  that  the  rate 
jumped  from  25  per  cent  in  4  hours  to  750  per  cent  in  2  hours.  The 
swelling  at  the  end  of  24  hours  was  1,778  per  cent,  at  which  time  in- 
crease was  still  in  progress.  The  total  increase  now  amounted  to  3,195 
per  cent.  The  unsatisfied  capacity  would  have  doubtless  carried  the 
swelling  to  a  thickness  as  great  as  any  yet  observed  in  any  of  the  bio- 
colloids  and  in  excess  of  the  sections  which  were  swelled  initially  in  the 
salt  alone. 

The  mass  was  now  nearly  97  per  cent  water.  Replacement  of  the 
water  by  acidified  salt  solution  was  followed  by  a  shrinkage  of  about 
200  per  cent  of  the  original  thickness  of  the  sections  in  8  hours.  When 
this  was  washed  out,  the  loss  was  regained  in  a  few  hours.  These 
sections  were  now  set  aside  for  desiccation.  The  final  period  of  16 
hours  in  the  swelling  was  in  distilled  water,  so  that  the  acidified  potas- 
sium nitrate  in  which  the  sections  had  been  immersed  for  the  preceding 
8  hours  must  have  been  reduced  to  a  very  small  amount. 

The  last  desiccation  had  left  the  sections  warped  to  some  extent, 
which  interfered  with  accurate  remeasurements,  but  the  thickness  had 
been  reduced  from  0.18  to  0.16  mm.  and  even  thinner.  The  irregulari- 
ties of  course  operated  to  introduce  some  error  in  the  swelling,  which 
would  make  the  total  appear  to  be  100  to  200  per  cent  less  than  it 
should  be. 

The  swelling  in  distilled  water  at  18°  to  20°  C.  now  progressed  over 
a  period  of  about  96  hours,  reaching  a  total  of  1,625  per  cent.  Both 
the  amount  of  the  total  and  the  length  of  time  necessary  to  reach  full 
capacity  would  indicate  that  the  sections  still  contained  some  of  the 
protein  or  its  derivatives  and  perhaps  some  of  the  salts.  Agar  alone 
would  attain  full  capacity  at  a  lesser  volume  and  hi  a  shorter  time. 

A  second  set  of  sections  of  agar  90  and  oat  protein  10  parts  were 
first  treated  with  acidified  potassium  nitrate,  in  which  the  swelling  was 
about  500  per  cent  (the  solution  being  freshly  made)  hi  16  hours.  This 


86  Hydration  and  Growth. 

was  now  washed  out  with  water  and  the  swelling,  which  had  been 
interrupted  in  the  previous  test,  was  allowed  to  go  on  for  147  hours. 
Swelling  was  still  in  progress  after  6  days  of  continuous  imbibition,  the 
increase  being  1,833  per  cent.  The  total  increase  up  to  this  time  was 
2,388  per  cent.  The  shrinkage  caused  by  a  solution  of  acidified  potas- 
sium nitrate  was  very  slight,  being  less  than  100  per  cent  of  the  origi- 
nal, and  was  scarcely  more  than  the  effect  of  the  potassium  nitrate 
which  was  first  applied.  The  original  thickness  of  0.18  mm.  was  now 
reduced  to  0.16  mm.,  much  of  this  loss  being  attributable  to  the  solu- 
tion out  of  some  of  the  water-soluble  oat  protein  and  some  of  the  agar 
(see  p.  107).  The  sections  also  probably  contained  some  salt  and 
probably  a  trace  of  acid. 

The  trio  of  sections  were  placed  under  the  auxograph  in  a  chamber 
where  the  temperature  of 'the  distilled  water  in  which  they  were  im- 
mersed varied  between  17°  to  20°  C.  in  the  6  days  during  which  the  test 
was  carried  on.  The  initial  swelling,  hi  contrast  with  the  action  of 
fresh  sections,  was  very  slow.  Furthermore,  the  increase  was  contin- 
ued over  a  long  period,  and  came  to  a  total  of  938  per  cent,  or  less  than 
one-half  of  the  original  expansion. 

Sections  of  plates  0.18  mm.  in  thickness  of  agar  90  parts  and  bean 
protein  10  parts,  to  which  0.85  per  cent  of  nutrient  salts  had  been 
added,  were  tested  in  the  chamber  at  16°  to  19°C.  The  initial  treat- 
ment with  distilled  water  induced  a  swelling  1,861  per  cent  in  20 
hours,  during  which  time  the  nutrient  salts  must  have  been  partially 
dissolved  out,  so  that  at  the  end  of  this  period  the  liquid  was  a  salt  solu- 
tion in  which  none  of  the  various  components  was  as  concentrated  as 
0.001  M,  since  about  30  c.c.  of  water  was  poured  in  the  dish.  The 
capacity  for  increase  in  this  solution  having  been  approached,  the  water 
(or  saline  solution)  was  replaced  with  a  hundredth-normal  solution  of 
malic  acid,  which  caused  a  shrinkage  of  about  100  per  cent  hi  2  or  3 
hours.  After  7  hours  the  acid  was  replaced  with  distilled  water,  and  a 
slow  swelling  ensued.  The  effects  of  the  alternations  here  are  not  sep- 
arable from  those  in  which  the  salt  is  incorporated  in  the  colloid  with 
the  first  swelling  solution. 

A  similar  set  of  sections  containing  nutrient  salts  were  prepared  by 
cutting  a  trio  of  strips  7  mm.  in  length  to  be  placed  under  the  auxo- 
graph. A  free  strip  30  mm.  long  was  also  placed  in  the  dish. 

The  initial  immersion  in  0.5  per  cent  nutrient  solution  was  practi- 
cally complete  as  to  its  swelling  effects  in  18  hours,  with  an  increase  of 
850  per  cent.  Substitution  of  hundredth-normal  acidified  potassium 
nitrate  caused  a  total  additional  increase  of  75  per  cent  in  22.5  hours. 
Replacement  of  this  solution  with  an  alkaline  solution  of  potassium 
nitrate  of  the  same  concentration  was  followed  by  a  further  swelling 
of  350  per  cent  in  44  hours.  The  total  swelling  of  the  sections  during 
a  period  of  110  hours  was  1,250  per  cent.  The  concentration  of  the 


Fluctuating  or  Alternating  Hydration  Effects,  87 

salts  was  reduced  during  one  period  of  23.5  hours  and  during  the  re- 
mainder of  the  time  the  concentration  ^was  about  a  hundredth  molar 
at  a  temperature  of  18°  to  21°  C.  No  appreciable  change  in  length  of 
the  tested  trio  of  sections  or  of  the  free  long  strip  occurred  (see  p.  19). 

The  effects  of  swamp  water,  which  has  already  been  shown  to  retard 
hydration,  were  tested  in  this  connection.  The  first  lot  of  water  of  this 
kind  was  procured  from  the  sedge-grass  swamps  near  Anoka,  Minnesota. 
Sections  of  agar  90  and  oat  protein  10  parts,  which  were  0.18  mm. 
in  thickness,  were  swelled  in  this  at  17°  to  19°  C.  Imbibition  was 
rapid  during  the  first  12  hours,  during  which  time  a  total  enlargement 
of  972  per  cent  took  place.  At  the  end  of  this  time  the  rate  had 
decreased  notably,  but  was  maintained  in  such  manner  that  in  4  days 
an  additional  swelling  of  361  per  cent  was  recorded.  The  volume 
was  now  practically  stationary  and  the  test  was  closed  at  the  end  of 
108  hours.  This  long-continued  swelling  at  a  low  rate  and  low  total 
was  in  contrast  with  swellings  of  similar  sections  in  distilled  water  and 
in  nutritive  salts. 

The  trio  of  sections  placed  in  distilled  water  at  the  same  time  as  the 
above  swelled  2,361  per  cent  in  3  days,  and  as  increase  had  ceased,  the 
distilled  water  was  replaced  with  swamp  water,  with  the  result  that  a 
slow  shrinkage  ensued,  which,  however,  amounted  to  only  36  per  cent 
of  the  original  volume  in  2  days. 

It  was  next  thought  important  to  test  the  swelling  of  sections  con- 
taining salts  in  swamp  water,  and  a  mixture  of  agar  90  parts,  bean 
protein  10  parts,  and  nutrient  salts  0.85,  0.18  mm.  in  thickness,  was 
swelled  at  the  temperatures  ofl7°tol9°C.  The  initial  swelling  was  prac- 
tically complete  in  16  hours,  at  which  tune  an  increase  of  1,082  per 
cent  had  taken  place.  Replacement  with  acidified  potassium  nitrate, 
hundredth  normal,  for  36  hours  caused  no  appreciable  change  in  vol- 
ume, but  when  this  solution  was  washed  away  in  swamp  water,  swell- 
ing was  resumed  and  a  further  increase  occurred,  most  of  which  took 
place  in  the  first  4  hours,  but  which  was  still  in  progress  at  a  very  slow 
rate  at  the  end  of  38  hours.  The  enlargement  in  the  swamp  water 
during  this  final  period  was  306  per  cent,  the  total  swelling  being 
1,388  per  cent. 

Sections  identical  with  the  above  were  first  placed  in  a  0.5  per  cent 
nutrient  solution,  in  which  a  swelling  of  888  per  cent  occurred  in 
17  hours,  at  which  time  the  pen  of  the  auxograph  was  tracing  a  hori- 
zontal line.  Replacement  with  acidified  potassium  nitrate  produced 
no  effect  beyond  a  very  slight  swelling.  When  the  acid  solution  was 
washed  away  with  the  swamp  water  swelling  began  and  an  increase 
amounting  to  333  per  cent  followed  in  40  hours.  This  final  action  was 
fairly  equivalent  to  that  of  the  preceding  series,  except  that  the  sub- 
stitution of  swamp  water  for  the  acidified  salt  was  followed  by  a  much 
slower  but  more  extended  rate  of  initial  swelling. 


88 


Hydration  and  Growth. 


Another  test  was  made  for  the  purpose  of  comparison  of  the  effects 
of  the  swamp  water  with  those  of  a  solution  of  nutrient  salts  in  which 
the  initial  swelling  in  the  nutrient  solution  was  861  per  cent  in  20 
hours,  which  was  less  than  in  swamp  water.  A  solution  of  acidified 
potassium  nitrate  was  substituted  and  a  swelling  of  55  per  cent  ensued 
in  55  hours.  Replacement  of  the  acidified  salt  solution  by  distilled 
water  was  followed  by  a  long-continued  swelling  which  increased  the 
volume  111  per  cent  in  40  hours.  The  total  increase  of  this  section 
was  thus  1,027  per  cent,  as  compared  with  1,333  per  cent  in  bog 
water  alone. 

The  obvious  importance  of  these  reactions  is  amply  illustrated  by 
the  changes  in  environmental  conditions  to  which  many  aquatic  plants 
are  subjected  in  the  course  of  a  season  or  even  in  a  day. 

Growth  in  all  probability  implies  the  incorporation  of  new  material 
in  the  colloid  which  adds  to  the  hydration  total  of  the  mass.  It  is  not 
easy  to  arrange  the  introduction  of  unhydrated  particles  in  a  swelling 
mass,  but  it  was  deemed  worth  while  to  bring  a  desiccated  trio  of  sec- 
tions into  swelling  under  a  similar  trio  which  had  already  undergone 
some  expansion. 

Three  sections  of  an  agar  90,  peptone  10  parts,  0.18  mm.  in  thick- 
ness, were  swelled  in  distilled  water  until  an  expansion  of  1,195  per 
cent  had  been  reached  in  4  hours;  a  second  series  of  sections  were  gently 
slid  under  the  first  lot,  the  pen  was  readjusted  to  register  continuously, 
and  a  fresh  lot  of  water  was  added  (fig.  14). 


I2p.m 


7T 

/                                      / 

1                                      i 

\  i 

1 

—  .                                  i 

\                                  j 

V 

\ 

rzs                   \ 

\ 

\ 

\                         \ 

FIG.  14. 

Course  of  swelling  of  a 
double  trio  of  sections 
of  agar  and  peptone, 
the  second  being  added 
after  the  first  was  par- 
tially hydrated.  See 
text,  page  88. 


A  swelling  of  the  two  trios  now  followed  which  reached  satisfaction 
in  about  14  hours,  which  amounted  to  708  per  cent,  calculated  on  the 
thickness  of  both.  Most  of  this  swelling,  however,  was  in  the  freshly 
added  sections,  in  which  the  swelling  was  1,083  per  cent  in  4  hours, 
and  a  lesser  amount  was  due  of  course  to  the  original  trio.  The  final 
total  of  the  pair  of  trios  swelled  only  1,153  per  cent,  while  the  first  set 
had  increased  1,195  per  cent  before  the  second  pair  was  added  and  had 
not  yet  completed  its  swelling.  It  seems  fair  to  infer  that  increase 
of  thickness  diminishes  the  proportionate  increase. 

A  second  test  was  made  in  which  the  swelling  of  the  first  trio  reached 
about  800  per  cent  in  2  hours  and  then  a  fresh  section  was  thrust  under 


Fluctuating  or  Alternating  Hydration  Effects. 


89 


one  of  the  old  ones.  The  swelling  during  the  next  two  and  a  half  hours 
was  equivalent  to  500  per  cent  on  the  basis  of  the  average  thickness 
of  the  four  sections  in  action. 

A  second  fresh  section  was  placed  under  another  old  one,  making 
five  sections  in  action.  The  swelling  during  the  next  3  hours  was 
something  over  300  per  cent,  calculated  on  the  basis  of  the  five  sections 
engaged.  The  third  fresh  section  was  now  added,  giving  a  preparation 
in  which  sections  were  included  in  four  different  stages  of  swelling,  with 
an  initial  average  thickness  of  0.36  mm.  The  swelling  during  the  next 
three  and  a  half  hours  was  about  225  per  cent  of  the  initial  thickness 
and  reached  an  end-point  20  hours  after  the  experiment  was  begun 
at  1,333  per  cent  of  the  total,  which  was  in  excess  of  that  reached  in  the 
previous  test.  The  temperature  underwent  a  range  of  from  15°  to 
22°  C.  (fig.  15). 


12  p.m. 


5 
15 
25 
35 
45 
55 
65 
75 
85 
95 
105 


FIG.  15. — Courses  of  swelling  of  sections  of  agar  and  peptone,  a  fourth,  fifth,  and  sixth  fresh  (dry) 

section  being  added  as  indicated. 

The  water  was  now  removed  from  the  dishes  and  the  double  trio  of 
sections  was  subjected  to  the  action  of  a  2  M  solution  of  calcium 
chloride.  A  shrinkage  followed  which  terminated  with  some  abrupt- 
ness at  the  end  of  an  hour  and  reduced  the  thickness  of  the  swelled 
sections  1.05  mm.  or  nearly  300  per  cent  of  the  original.  Calculated 
on  the  basis  of  the  swelled  sections,  which  had  reached  a  total  average 


55 
65 
75 

8am                  in                                                                             12  pm. 

"1-1                                                                                 '  " 

/         ' 

X-  _\  \  

FIG.  16. — Variations  in  volume  of  double  trio  of  sections  of  agar  and  peptone  which  had  reached 
full  hydration  in  distilled  water  and  were  then  immersed  in  calcium  chloride,  2M. 

thickness  of  4.8  mm.,  the  reduction  was  nearly  22  per  cent.  A  swell- 
ing now  followed  and  practically  half  of  the  thickness  lost  was  regained. 
Most  of  this  was  completed  within  2  hours,  after  which  a  very  slow 
rate  of  increase  followed,  which  was  not  at  an^end  at  the  close  of  the 
third  day.  This  test  also  was  carried  on  at  room  temperature,  which 
underwent  a  wide  variation,  as  noted  above  (fig.  16). 


Hydration  and  Growth. 


These  elementary  experiments  open  a  field  of  possibilities  as  to  the 
incorporation  of  new  material  in  masses  of  swelling  colloids,  in  exempli- 
fication of  some  phases  of  cell-mechanics,  inclusive  of  the  action  of 
embryonic  cells  in  growing  regions.  The  author,  in  collaboration  with 
Spoehr  and  Richards,  has  recently  been  able  to  outline  the  manner 
in  which  the  xerophilous  and  the  succulent  types  of  shoots  or  organs 
are  due  to  the  action  of  such  superposed  effects  in  the  cell  colloids. 
A  series  of  detailed  analyses  of  the  carbohydrate-content  of  the  opun- 
tias,  which  was  arranged  to  determine  the  principal  sugars  not  only 
during  developmental  stages,  but  also  to  follow  the  changes  through- 
out the  seasons,  established  the  fact  that  when  these  plants  were  sub- 
subjected  to  long  periods  of  drought,  resulting  in  partial  desiccation, 
polysaccharids  were  dehydrated,  with  the  result  that  the  sugars,  which 
have  a  low  water  capacity,  became  converted  into  pentosans  or  muci- 
lages, which  have  a  large  imbibition  capacity.  This  fact  was  amply 
confirmed  by  the  coefficients  of  swelling  which  were  obtained  in  my 
own  tests,  which  ran  through  several  years. 

Following  this,  the  fortunate  discovery  was  made  that  Castilleia 
latifolia,  which  is  native  to  the  region  about  the  Coastal  Laboratory, 
has  thin,  highly  acid  leaves  when  growing  under  mesophytic  conditions, 
but  has  less-acid  succulent  leaves  in  arid  locations,  the  increased  size 
of  the  leaves  being  due  to  the  hypertrophy  of  the  thin-walled  paren- 
chymatous  tissues.  The  hydration  reactions  of  the  two  types  of 
leaves  in  a  fresh  and  dried  condition  are  shown  in  table  70,  which  gives 
the  swellings  in  0.01  normal  citric  acid  at  15°  C. 

TABLE  70. 


Thin. 

Succulent. 

Thickness. 

Swelling. 

Thickness. 

Swelling. 

Fresh  leaves  

mm. 
/  0.4 
I     -41 

p.  ct. 
125 
184 

mm. 
1.4 
1.4 

p.  ct. 
21 
25 

Above  leaves  dried  and  re-hydrated  

/     .23 
\     .25 

42 
20 

0.5-0.6 
0.63 

95 
91 

(Expansion  in  terms  of  dried  thickness.) 
Fresh-dried  leaves  

{     .38 
•2 
|      .38 
I      -2 

1.2 
.5 
1.1 

.38 

25 

120 

(Expansion  in  terms  of  dried  thickness.) 

62 

92 

In  the  interpretation  of  these  results,  it  is  to  be  noted  that  drying, 
both  from  the  fresh  state  and  from  the  hydrated  condition,  reduces  the 
hydration  capacity  of  the  thin  leaves,  but  not  of  the  succulent  ones. 
The  principal  changes  in  hydration  include  the  extraction  of  acids  and 
salts  as  well  as  the  hexoses,  while  the  mucilaginous  pentosans  diffuse 


Fluctuating  or  Alternating  Hydration  Effects.  91 

not  at  all  or  very  slowly.  The  swelling  of  the  succulent  leaves  is 
therefore  high  after  extraction  by  reason  of  the  presence  of  these  sub- 
stances, which  have  been  partially  freed  from  the  retarding  action  of 
the  organic  acids  and  salts.1 

The  conversion  of  the  diffusible  sugars  to  the  mucilaginous  pentosans 
is  one  of  the  alterations  which  may  result  in  the  cell  as  a  result  of  partial 
desiccation,  with  a  striking  morphogenetic  result  as  indicated.  Under 
certain  conditions  of  desiccation,  the  lessening  of  the  water-content  of 
the  cell  accelerates  the  formation  of  the  anhydrides  of  which  wall 
material  is  composed,  with  the  result  that  the  hydration  capacity  of 
the  product  in  this  case  is  less  than  that  of  the  polysaccharids,  and  in 
addition  to  this  direct  diminution  of  the  water-absorbing  material  of 
the  cell,  the  production  of  the  heavy  wall  at  an  early  stage  of  the 
development  of  the  cell  checks  growth,  resulting  in  the  restricted 
shoots  or  organs  with  indurated  membranes  which  are  characteristic 
of  xerophytic  plants.2  Two  striking  and  highly  important  vegetational 
types  are  thus  seen  to  result  more  or  less  directly  from  the  action  of  the 
environment  upon  the  cell-colloids,  hi  accordance  with  a  view  expressed 
by  the  author  in  a  previous  publication. 

1  MacDougal,  Richards,  and  Spoehr.     The  basis  of  succulence  in  plants.     Bot.  Gaz.,  67:  405 
1919. 

2  MacDougal,  D.  T.     The  Salton  Sea,  etc.     Carnegie  Inst.  Wash.  Pub.  No.  193.     1914.     See 
p.  179.     Also  MacDougal  and  Spoehr.    The  origination  of  xerophytism.    Plant  World,  21:  245. 
1918. 


VIII.  WATER  DEFICIT,  OR  UNSATISFIED  HYDRATION 

CAPACITY. 

Much  of  the  present  confusion  as  to  the  nature,  mechanism,  and 
course  of  growth  is  due  to  the  fact  that  premature  attempts  have  been 
made  to  institute  comparisons  in  measurements  derived  from  organ- 
isms fundamentally  different. 

Thus,  the  growth  of  bacteria  consists  in  the  enlargement  to  a  unit 
size  of  cells  high  in  proteins  which  become  independent  when  this 
volume  is  reached,  and  not  being  attached  to  other  cells,  their  presence 
does  not  directly  affect  the  rate  of  growth  calculated  upon  their  num- 
ber, except  in  so  far  as  their  excretions  hi  the  medium  may  do  so. 
Furthermore,  these,  as  well  as  organs  which  are  submerged  hi  the 
liquid  nutrient  media,  are  in  a  condition  approaching  complete  hydra- 
tion  in  the  complex  of  conditions  in  which  they  live,  and  these  may 
vary  only  within  very  narrow  limits  in  many  cases. 

Growth  in  the  higher  complex  plants  implies  the  multiplication  of 
embryonic  cells  and  the  development  of  the  greater  number  of  them 
into  special  static  tissues  to  which  the  growing  cells  are  inseparably 
attached.  Thus  an  internode,  or  a  leaf,  barely  makes  its  appearance 
before  some  of  its  cells  have  passed  beyond  the  growing-stage  and  into 
a  condition  of  lessening  change  to  a  nearly  static  condition  of  maturity. 
Enormous  numbers  of  senescent  and  highly  specialized  cells  are  formed 
through  which  the  water-supply  of  the  growing  cells  must  pass  and 
which  compete  for  the  supply.  The  water-deficit,  or  the  amount  which 
a  cell-mass  hi  the  plant  may  take  up,  may  therefore  vary  widely,  as, 
in  addition  to  the  internal  changes,  water  is  constantly  being  lost  from 
the  surfaces  by  transpiration.  An  instrument  attached  to  the  terminal 
portion  of  a  plant  records  the  changes  hi  volume  of  cell-masses  in  all 
of  the  possible  stages  between  the  apex  and  the  base  of  the  internode 
or  the  point  at  which  the  stem  may  be  fixed  in  the  experimental  pre- 
parations. 

The  tips  of  roots  offer  a  generalized  type  of  growth,  which  has  been 
the  subject  of  more  experimentation  than  any  other  part  of  the  higher 
plants.  Even  here  the  measurements  invariably  include  the  enlarge- 
ments of  masses  of  embryonic  cells  by  imbibition  alone,  the  inter- 
mediate stage  in  which  the  increase  in  the  size  of  the  vacuole  doubtless 
plays  an  important  r61e,  to  a  final  stage  in  which  imbibition  again 
may  be  the  only  force  of  distension.  Only  in  individual  unicellular 
organisms  and  hi  such  structures  as  pollen-tubes  may  growth-enlarge- 
ments be  dealt  with  to  the  exclusion  of  variations  of  mature  tissues. 

Again,  growth  is  a  resultant  of  the  play  of  molecular  forces  in  sur- 
face tensions  and  of  a  series  of  metabolic  transformations,  hi  which 
features  notable  differentiations  are  found  in  the  groups  of  organisms. 

92 


Water  Deficit  or  Unsatisfied  Hydration  Capacity.  93 

The  main  features  of  imbibition  are  determined  by  the  carbohydrate- 
protein  ratio  and  the  presence  of  salts.  Animals  and  some  vegetal 
organisms  are  high  in  proteins.  The  vacuole  and  the  external  layer 
of  the  protoplasmic  units  in  plants  give  a  play  of  osmotic  activities 
not  duplicated  in  the  animal;  and  lastly,  as  has  been  so  clearly  estab- 
lished by  the  work  of  Dr.  H.  A.  Spoehr,  the  plant  has  a  characteristic 
carbohydrate  metabolism  with  the  capacity  for  the  synthesis  and  com- 
bination of  the  amino-acids,  while  in  animal  processes  metabolism  is 
more  largely  concerned  with  the  proteins,  and  amino-acids  result 
chiefly  or  entirely  by  disintegration  of  albumins. 

The  ordinary  conception  of  growth  implying  the  changes  in  dimen- 
sions of  the  developing  parts  of  organs,  shoots,  roots,  etc.,  is,  however, 
a  well-rounded  one  of  sound  value  and  worthy  of  attention  as  a  uni- 
fied procedure.  The  ultimate  physical  forces  concerned  are  those 
which  find  play  in  elastic  gels,  and  the  interaction  of  these  forces  may 
be  modified  by  the  self-altered  composition  of  the  living  colloids  and 
by  the  action  of  external  agencies  under  the  influence  of  which  these 
colloids  operate. 

The  course  of  growth  in  the  succulents  will  come  in  for  a  large  share 
of  attention  in  the  following  chapters.  The  form  of  the  organs  of 
such  plants  facilitates  the  making  of  observations  in  which  the  actual 
temperature  of  the  living  mass  may  be  found  with  some  exactness, 
and  the  large  bulk  which  characterizes  them  makes  it  comparatively 
easy  to  obtain  analyses  showing  the  varying  proportions  of  the  con- 
stituents of  importance  in  growth.  Some  attention  has  been  given 
to  plants  with  thin  stems  and  slender  leaves,  which  in  reality  constitute 
the  dominant  vegetative  type  of  plants. 

The  arrangement  of  imbibition  tests  of  the  growing  terminal  inter- 
nodes  of  stems,  and  other  organs,  and  the  interpretation  of  the  results 
was  made  upon  the  basis  of  the  supposition  that  such  material  was  in 
effect  a  complicated  salted  colloid,  with  an  altering  and  unsatisfied 
hydration  capacity.  The  manner  in  which  saturation  might  be 
reached  by  immersion  in  various  solutions  might  well  offer  some 
profitable  comparisons  with  the  behavior  of  biocolloids  of  known 
composition  and  structure.  The  earlier  trials  were  made  with  the 
growing  parts  of  stems  of  Phytolacca  decandra,  Micrampelis  calif  arnica, 
and  Rudbeckia  laciniata.  The  young  internodes  furnished  sections 
about  1  cm.  long  and  of  a  thickness  from  4  to  8  mm.  A  tangential 
slice  was  cut  from  one  side  to  remove  a  segment  of  the  fibrovascular 
tissue,  and  the  plane  surface  exposed  served  to  seat  the  sections  firmly 
in  the  glass  testing-dishes  of  the  auxographs.  The  first  series  was 
made  with  distilled  water,  sodium  or  potassium  hydroxide,  and  citric 
and  formic  acids,  all  in  hundredth-normal  concentration. 

The  series  which  were  run  in  a  day-lighted  laboratory  at  tempera- 
tures of  18°  to  22°  C.  agreed  in  swelling  most  in  distilled  water,  less  in 


94 


Hydration  and  Growth. 


41.9 


the  hydroxid,  and  least  in  the  organic  acids  mentioned,  in  propor- 
tions of  40,  20,  and  15  in  Rudbeckia,  and  30,  25,  and  15  in  Micrampelis. 
Phytolacca  gave  proportions  of  30,  25,  and  12  under  the  same  circum- 
stances. It  was  noticeable  that  distension  due  to  swelling  by  these 
acids  was  relaxed  after  a  few  hours,  probably  due  to  the  solution  out 
of  some  of  the  colloidal  contents  of  the  cells. 

Further  tests  were  made  with  the  stalks  of  Rudbeckia  bearing  heads 
of  flowers  about  ready  to  open.     The  greater  part  of  the  total  growth 
had  been  accomplished,  although  they  were  still  in  rapid  action  and 
the  material  for  the  developing  flowers  was  being  drawn  from  them. 
Tangential  slices  less  than   1  mm.  in  thickness  were  removed,  leav- 
ing the  stalks  4  mm.  in   thickness  at 
the  larger  end  and  about  3.4  mm.  at 
the  smaller.      Sections  about  1  cm.  hi 
length  were  cut  from  four  such  stalks 
and  the  whole  number  was  divided  into 
two  lots.     One  lot  of  sections  was  taken 
into   the   dark   room    and    swelled   at 
18°  C.   and  the  others  were  set   on   a 
window-ledge  to  become  air-dry.    The 
average  thickness  was  taken  to  be  3.7 
mm.     Twenty  hours  later  swellings  of 
6.8  per  cent  in  distilled  water,  4.8  per 
cent  in  alkali,  and  1.4  per  cent  in  acid 
had  been  made  (fig.  17).     Actual   en- 
largement by  imbibition  in  the  acid  had 
lasted  only  about  an  hour,  after  which 
a   shrinkage  ensued   that  reduced  the 
thickness  5.4  per  cent  from  the  original 
volume  and  6.8  per  cent  from  the  max- 
imum.   The  decrease  here  and  the  in- 
crease in  water  and  in  hydroxid   were 
all  in  progress  at  the  close  of  the  test. 
The  dried  sections  came  down  to  about 
half   of   their   original  diameter,    but,  on  account  of  the  irregular 
shapes  assumed,  could  not  be  measured  with  accuracy.    Trios  of  these 
under  the  auxograph  gave  swellings  of  41.9  per  cent  in  distilled  water, 
21.6  per  cent  in  hundredth-normal  citric  acid,  and  37.8  per  cent  in 
hundredth-normal  sodium   hydroxid.     The  sections  were  thus  seen 
to  return  to  about  their  original  size  in  the  living  condition  in  dis- 
tilled water — nearly  this  size  in  hydroxid,  but  far  short  of   it  hi 
the  acid  solution,  after  the  manner  of  a  salted  biocolloid  consisting 
of  a  large  proportion  of  carbohydrate  and  a  smaller  one  of  protein. 
Here,  as  in  all  comparisons  between  the  hydration  of  living  and  of 
dried  sections,  it  must  be  taken  into  account  that  in  the  desiccation 


Dried 


Fresh 


6.8 


37.8 


sections 


material 
4.8 


21.6 


1.4 

Water  Alkali  Acid 

FIG.  17. — Swelling  reactions  of  fresh 
and  dried  flower-stalks  of  Rud- 
beckia, in  percentages  of  original 
thickness.  The  proportionate  in- 
crease of  dried  sections  is  denoted 
by  the  heights  of  the  vertical  lines, 
and  that  of  fresh  sections  by  the 
marks  near  base. 


Water  Deficit  or  Unsatisfied  Hydration  Capacity. 


Young. 

Mature. 

Dried. 

Water. 

p.  ct. 
40 

p.  ct. 
68 

p.  ct. 
419 

Acid  

68 

14 

48 

Hydroxid  .  .  . 

419 

216 

378 

of  cell-masses,  the  acids,  salts,  sugars,  etc.,  in  the  vacuoles  and 
syncretic  cavities  of  the  protoplasm  increase  in  concentration  as  the 
dehydration  proceeds,  until  as  the  end  is  neared  these  substances  are 
taken  up  by  the  protoplasmic  colloids  from  solutions  which  may  be 
saturated. 

The  swelling  of  a  living  section  continues  until  hydration  capacity 
is  satisfied  or  balanced  against  the  mechanical  restraint  of  the  cell- 
walls,  and  includes  the  possibilities  of  osmotic  action  by  reason  of  their 
differential  action.  The  colloids  are  subjected  to  the  action  of  the 
salts  and  acids  only  to  attenuations  in  which  these  substances  are 
usually  present.  When  desiccated  sections  are  swelled,  the  cell  col- 
loids are  now  in  the  condition  of  having  adsorbed  substances  previously 
hi  great  dispersion  and  the  external  layers  of  the  cells  now  exercise 
only  the  effect  of  a  dense  filter- 
paper;  consequently  any  osmotic  TABLE  H-*"**  «/  Rudbeckia. 
pressure  which  might  be  set  up 
is  soon  equalized,  and  hence  con- 
tributes but  little  to  the  ex- 
pansion of  the  cells. 

Returning  to  the  matter  of  the 
fresh  sections  of  Rudbeckia,  the 
nature  of  the  increase  in  hydra- 
tion capacity  of  the  material  with  the  march  of  development  may  be 
set  forth  more  clearly  by  the  figures  in  table  71 ,  in  which  the  percent- 
ages of  swelling  are  multiplied  by  10. 

Swelling  was  greatest  in  water  in  both  young  and  old  stalks  and  the 
increase  of  the  hydrogen-ion  concentration  or  acidity  of  the  medium 
resulted  in  reducing  the  final  hydration  capacity  of  the  tissues,  more 
water  being  taken  up  when  hydroxid  was  used,  but  the  total 
capacity  being  less  than  in  water.  The  desiccation  of  the  stems, 
with  attendant  possible  coagulatory  effects,  was  accompanied  by  an 
increase  in  hydration  capacity  in  the  acid  solutions.  The  greater 
swelling  shown  by  old  tissues  suggests  increased  pentosan  or  mucil- 
age content  rather  than  a  deficit  due  to  loss  of  water. 

No  opportunity  was  lost  to  make  measurements  of  other  material 
which  might  possibly  show  the  diversity  of  action  of  different  plants. 
The  terminal  internodes  of  Verbena  ciliata  were  available  at  the  Desert 
Laboratory  in  March  1918,  and  sections  of  these  with  a  tangential 
slice  removed  were  about  1.8  mm.  in  thickness.  Swellings  at  18°  to 
20°  C.  were  as  shown  in  table  72. 

TABLE  72. 

p.  ct. 

Distilled  water 7 

Citric  acid,  0.01  N 2.8 

Sodium  hydroxid,  0.01  M 7 

Potassium  chloride,  hydrochloric  acid,  0.01  M 4 


96  Hydraiion  and  Growth. 

The  familiar  relation  much  seen  in  young  organs,  by  which  the 
imbibition  is  least  in  the  acid  and  most  in  water  and  alkaline  solutions, 
is  exhibited.  Some  interest  attaches  to  this  plant  from  the  fact  that 
its  expressed  and  centrifuged  juice  has  been  found  by  Dr.  H.  A.  Spoehr 
to  aggregate  and  form  a  fairly  firm  jelly  without  concentration,  an 
action  probably  due  to  the  large  proportion  of  pentosans  present. 

Forms  of  Brodicea  native  to  Tumamoc  Hill  start  into  activity  late 
hi  February  and  by  the  end  of  the  first  week  in  March  have  formed  new 
conns  on  the  crowns  of  the  older  ones  nearly  1  cm.  in  diameter,  from 
the  apices  of  which  the  long,  slender  leaves  extend  at  a  rapid  rate. 
Halves  of  these  corms  arranged  in  the  dishes  under  the  auxographs 
gave  swellings  at  ,20°  C.  as  shown  in  table  73. 

TABLE  73. 

p.  ct. 

Distilled  water 5 

Citric  acid,  0.01  N 2.7 

Sodium  hydroxid,  0.01  M  (not  completed) 2.2 

Potassium  chloride,  hydrochloric  acid,  0.01  M 2.5 

The  carbohydrate  material  in  these  organs  is  starch,  and  this  and 
the  products  of  its  hydrolysis  constitute  the  main  components  of  the 
cells.  The  increases  noted  above  are  low,  that  in  the  alkaline  solution 
being  still  in  progress.  The  old  corms  form  tapering  extensions  from 
the  lower  surf  ace/ which  hi  the  earlier  stages  are  made  up  only  of  thin- 
walled  cells,  filled  with  the  products  of  starch  hydrolysis.  These 
show  swellings  of  17  per  cent  hi  distilled  water  at  the  above  tem- 
perature. Two  other  variations  were  tried  to  ascertain  whether  the 
young  corm  just  formed,  having  swelling  capacities  as  above,  differed 
in  hydration  capacity  from  the  older  basal  corms  being  emptied  of  their 
contents.  This  was  accomplished  by  setting  up  a  preparation  of  three 
plants  in  which  the  young  corms  were  seated  in  place  on  the  older 
conns.  The  average  height  of  the  preparation  was  12  mm.  and  the 
increase  in  distilled  water  was  9.6  per  cent  at  20°  C.  Another  set  of  prep- 
arations was  made,  in  which  older  corms  from  which  the  young  portion 
had  been  removed  were  immersed  in  water  at  similar  temperatures. 
The  increase  in  this  case  was  24  per  cent,  showing  that  these  structures, 
hi  which  the  accumulated  starch  was  hi  an  advanced  stage  of  hydrolysis, 
had  an  imbibition  capacity  much  greater  than  that  of  the  young  corms. 
The  fact  must  be  taken  into  account  that  the  young  corms  were  firm 
and  solid  to  the  touch  and  were  to  be  regarded  as  in  a  high  state  of 
imbibition,  while  the  old  corms  had  been  partially  emptied,  but  were 
capable  of  returning  to  the  dimensions  of  their  original  turgid  con- 
dition. 

Tangential  slices  from  the  terminal  internodes  of  asparagus  tips, 
bought  hi  the  market  in  Tucson  in  March,  were  made  to  have  a  thick- 
ness of  about  3  mm.,  and  these  gave  swellings  as  shown  in  table  74. 


Water  Deficit  or  Unsatisfied  Hydration  Capacity. 


97 


TABLE  74. 

p.ct. 

Distilled  water 7 

Citric  acid,  0.01  N 4 

Sodium  hydroxid,  0.01  M 6.2 

Potassium  chloride,  hydrochloric  acid,  0.01  M.    Immediate  and  marked  shrinkage. 

The  rapid  and  immediate  shrinkage  of  these  thin  sections  hi  the 
acidified  salt  solution  was  so  striking  that  a  second  measurement  was 
made,  in  which  thicker  sections  swelled  4.4  per  cent  hi  the  acidified 
salt  solution  within  a  few  minutes  and  then  began  to  shrink  to  the 
original  dimensions  and  to  smaller  volume.  Imbibition  hi  the  salt  solu- 
tion alone  showed  two  phases :  first,  a  very  rapid  swelling  to  a  volume 
near  the  maximum  capacity,  then  a  slow  increase  which  was  still  hi 
progress  24  hours  later  and  which  at  that  time  had  brought  the  section 
to  a  thickness  10  per  cent  greater  than  the  original.  Such  material  hi 
acids  swells  quickly  and  more  gradually  than  in  the  combined  solu- 
tion, then  slowly  shrinks  to  the  original  dimensions  and  below.  The 
presence  of  the  salt  accentuates  the  action  of  the  acid,  as  it  did  also  in 
the  swelling  of  stems  of  Verbena  hi  similar  solutions,  the  increase  hi  the 
acid  being  but  2.8  per  cent,  while  it  was  4  per  cent  hi  the  combined  solu- 
tion, the  explanation  of  which  is  probably  to  be  sought  hi  the  com- 
bined effects  of  acids  and  bases  (fig.  18). 


8a.m  9     10      II      m.    lp.m.2       345     6p.m.7       8      9      10     II    I2p.mlain.2       3. 

,7  A—  A  —  A—-/-  -/—  -1—  /-  -/     /  -V  --A  -A—  A    -^--V     A    -/—  V   -A—  /- 

*2     111    il    r  T    T    1    irrilfllri 

////////////  _--/-    /     /     /     /    /     /     /     i 

\  

/—  /—  4—  I— 

I-H- 

ri 

1  1 

1 

1 

r, 

d 

.-'" 

FIG.  18. — Swelling  of  tangential  slices  from  tips  of  asparagus  shoots  at  14°  to  17°  C.,  X  20,  on 
scale  ruled  to  5  mm.  and  1-hour  intervals,  a,  swelling  of  trio  3.8  mm.  in  thickness  7  per 
cent  in  water;  b,  swelling  of  trio  3.7  mm.  in  thickness  4  per  cent  in  hundredth-normal  citric 
acid;  complete  within  a  few  minutes,  followed  by  gradual  shrinkage;  c,  swelling  of  trio  of 
sections  6.2  per  cent  in  sodium  hydroxid  with  final  shrinkage;  d,  slight  swelling  of  trio  of 
sections  in  hundredth-normal  potassium  chloride  and  hydrochloric  acid  and  then  rapid 
shrinkage. 

Other  aspects  of  the  matter  of  permeability  and  swelling  are  offered 
by  the  reactions  of  young  bean  seeds  which  had  not  attained  more  than 
an  eighth  of  their  final  volume  and  had  a  diameter  of  2.8  mm.  These 
were  tested  hi  a  series  parallel  to  the  above  and  swelled  as  follows: 


TABLE  75. 

p.ct. 

Distilled  water 8.2 

Potassium  nitrate,  0.01  M 9.9 

Potassium  nitrate,  citric  acid,  0.01  N 8.2 

Citric  acid,  0.01  N 5.4 

Potassium  hydroxid,  0.01  M 12.5 

The  proportions  here  were  also  those  of  an  agar-albumin  mixture, 
but  the  high  swelling  hi  the  acidified  salt  was  not  shown.    The  beans 


98  Hydration  and  Growth. 

were  intact,  with  an  unbroken  coat.  It  was  probable,  therefore,  that 
osmosis  may  have  played  some  part  in  the  absorption  in  water  and  the 
solutions. 

A  second  lot  of  the  growing  beans,  which  had  an  average  diameter 
of  2.4  mm.,  was  allowed  to  desiccate  for  3  days,  and  when  they  had 
become  hard  and  dry  and  were  dead,  the  swelling  tests  were  made 
with  them,  yielding  the  f  olio  wing  results : 

TABLE  76. 

p.  cL 

Distilled  water 29.2 

Potassium  nitrate,  0.01  M 22.9 

Potassium  nitrate,  citric  acid,  0.01  N 32 . 5 

Citric  acid,  0.01  N 29.2 

Potassium  nitrate,  potassium  hydroxid,  0.01  M 31 .3 

Potassium  hydroxid,  0.01  M 15.8 

It  is  evident  that  the  activity  of  the  vacuole  is  not  the  determining 
or  dominating  factor  in  the  water-deficit  or  unsatisfied  hydration 
capacity;  if  it  were,  the  greatest  swelling  would  have  taken  place  in 
water.  The  action  of  the  salt  was  not  increased  by  the  addition  of 
acid  in  the  swelling  of  the  living  cells,  but  such  an  effect  was  produced 
in  the  dried  beans.  The  interferences  were  much  less  in  the  driejl 
material,  the  swelling  in  acid  being  but  little  short  of  that  in  water, 
while  it  was  relatively  much  less  in  the  living  material. 

Young  leaves  of  Abronia  latifolia  which  had  been  allowed  to  wilt  for 
two  days  while  attached  to  stems  in  a  ventilated  room  were  cut  into 
suitable  strips  free  from  the  midrib  and  the  larger  veins  in  preparation 
for  swelling  tests.  The  spongy  texture  made  it  impossible  to  measure 
thickness  with  accuracy,  but  this  was  estimated  as  1  mm.  Increases 
in  thickness  were  measured,  as  follows: 

TABLE  77. 

p.  ct. 

Distilled  water 40 

Potassium  nitrate,  0.01  M 80 

Potassium  nitrate,  citric  acid,  0.01  M 55 

Citric  acid,  0.01  N 50 

Potassium  nitrate,  potassium  hydroxid,  0.01  M 50 

Potassium  hydroxid,  0.01  M 60 

Potassium  nitrate  gave  a  maximum  swelling,  a  lesser  one  when  acid 
was  added  with  the  salt,  and  still  less  swelling  ensued  when  acid  alone 
was  used.  The  swelling  in  distilled  water  probably  represents  a  nor- 
mal imbibition  total  under  the  undisturbed  conditions  in  the  leaf. 
The  partial  neutralization  of  the  acid  by  the  addition  of  hydroxid 
gave  a  swelling  determined  by  the  acidified  salts  formed. 

Half-grown  succulent  leaves  of  Cakile  sp.  were  taken  from  the  beach 
at  Carmel,  August  23,  1917.  The  total  acidity,  as  determined  by  Pro- 
fessor H.  M.  Richards,  was  found  to  be  equivalent  to  0.30  c.c.  hun- 
dredth-normal sodium  hydroxid  per  gram  of  fresh  material.  The 
strips  of  leaf-blades,  which  were  cut  in  such  manner  as  not  to  include 
any  of  the  main  veins,  had  an  average  thickness  of  1.2  mm.  One  lot 


Water  Deficit  or  Unsatisfied  Hydration  Capacity. 


99 


was  immersed  in  solutions  in  a  fresh  condition,  and  others  were  allowed 
to  lie  on  a  window  ledge  for  24  hours,  at  the  end  of  which  time  they 
were  dead,  shrunken,  but  limp,  so  that  they  remained  in  any  shape 
into  which  they  were  bent  or  twisted.  The  swellings  were  as  follows, 
on  the  basis  of  the  original  thickness : 


TABLE  78. 


Living. 

Dried. 

Distilled  water  

p.  ct. 
12.5 

p.  ct. 
20  8 

Potassium  nitrate,  0.01  M  

16.6 

25 

Potassium  nitrate,  citric  acid,  0.01  N  

16.6 

4.2 

Citric  acid,  0.01  N  

8.3 

16  6 

Potassium  nitrate,  potassium  hydroxid,  0.01  M 
Potassium  hydroxid,  0.01  M  

12.5 
16.6 

37.5 
33.3 

The  swelling  of  the  living  tissues  was  greatest  and  was  equal  in  salt, 
acidified  salt,  and  hydroxid,  while  it  was  low  in  acid.  The  dried 
material  swelled  most  in  the  salt,  while  it  was  least  in  acidified 
salt.  Increases  occurred  in  all  the  other  solutions,  the  swelling  in 
alkaline  salt  being  three  times  as  great  as  hi  the  living  material. 
It  was  evidently  desirable  to  measure  the  water-relations  of  some 
simple  plant  structure  which  could  be  brought  into  the  tests  with- 
out anatomical  injury  and  which  would  not  suffer  material  and 
immediate  injury  by  submersion.  The  small  tubers  of  the  potato 
seemed  to  meet  these  requirements. 

A  number  of  tubers  of  the  second  generation  of  a  hybrid  between  a 
domesticated  potato  and  Solarium  fendleri  of  Arizona  were  available, 
and  as  these  bodies  were  in  a  condition  in  which  they  were  ready  for 
planting  and  sprouting,  tests  were  arranged  to  obtain  the  swelling 
measurements  in  various  solutions.  Trios  of  tubers  5  to  8  mm.  hi 
diameter  were  placed  in  the  dishes  after  the  total  and  average  diameters 
had  been  ascertained.  The  first  set  swelled  7.5  per  cent  in  5  days  hi 
distilled  water  at  14°  to  21°  C.,  and  the  second  increased  the  same 
proportion  in  hundredth-normal  citric  acid  in  4  days.  A  series  ar- 
ranged to  obtain  the  auxographic  record  as  complete  as  possible  was 
allowed  to  run  for  11  days,  at  the  end  of  which  time  the  swelling  in 
water  amounted  to  13  per  cent,  in  hundredth-normal  citric  and 
hydrochloric  acids  14  per  cent,  and  less  than  7  per  cent  in  hydroxid, 
at  temperatures  between  14°  and  20°  C.  identical  for  the  lot.  Actual 
shrinkage  had  not  begun  in  any  of  the  solutions  and  all  solutions  were 
renewed  three  or  four  times  during  the  period. 

A  set  of  three  with  an  average  diameter  of  8.3  mm.  was  placed  in  a 
solution  of  calcium  chloride  3  N  acidified  to  0.01  N  with  hydrochloric 
acid.  A  steady  shrinkage  amounting  to  3.4  per  cent  in  4  days  began 
at  once. 


100  Hydration  and  Growth. 

Later,  when  the  tubers  had  begun  to  sprout,  a  set  was  arranged  to 
obtain  another  series  of  tests,  extended  over  a  period  of  22  days,  with 
many  renewals  of  the  solutions.  The  swellings  were  as  follows  at 
20°  C: 

TABLE  79. 

P.  ct. 

Distilled  water 34 . 4 

Citric  acid,  0.01  N 24.4 

Sodium  hydroxid,  0.01  M 13 . 8 

Potassium  chloride,  hydrochloric  acid,  0.01  M 20 

All  of  the  tubers,  with  two  exceptions,  were  turgid  and  firm  at  the 
end  of  the  tests,  even  when  extended  over  22  days.  Immersion  in 
any  of  the  solutions  for  a  day  or  two  usually  prevented  sprouting  by 
killing  the  buds,  although  the  remainder  of  the  tuber  kept  alive.  One 
of  the  three  tubers  in  water  in  the  last  test  sprouted  in  the  dish  at  the 
end  of  a  week  and  the  excessive  increase  shown  in  this  lot  may  be 
attributed  in  part  to  the  action  of  the  continued  hydrolysis  of  starch 
as  the  growth  of  the  etiolated  stem  proceeded. 

Stiles  and  J0rgensen  have  made  an  extensive  series  of  tests  of  the 
swelling  of  the  potato,  in  which  a  wholly  different  technique  was  used. 
Plugs  were  cut  from  tubers,  from  which  slices  2  mm.  in  thickness  were 
taken,  and  these  were  immersed  in  solutions  in  bottles.  Variations 
were  taken  by  weight  and  the  unavoidable  difficulties  of  the  method 
were  dealt  with  in  an  exact  manner.  The  interpretations  of  the  action 
of  the  sections  in  the  different  solutions  by  Stiles  and  J0rgensen  are 
all  based  on  the  assumption  that  the  hydration  is  one  based  entirely 
on  osmosis  and  that  swelling  ceases  when  the  parenchymatou?  walls 
become  permeable.1 

The  conditions  presented  by  my  experiments  included  the  action  of 
the  external  coat  of  the  tuber,  which  is  composed  of  non-living  elements 
the  permeability  of  which  to  water  has  been  tested  in  a  careful  manner 
in  certain  structures  by  Denny.  The  fact  that  the  swelling  was 
greater  in  water  than  hi  any  of  the  solutions  might  lead  to  the  con- 
clusion that  absorption  was  largely  by  osmosis.  Such  an  explanation 
can  not  be  accepted  as  an  adequate  one,  however.  The  cell  colloids 
of  the  potato,  being  high  in  carbohydrates,  would  show  the  greatest 
swelling  hi  water,  and  hydration  would  be  retarded  and  limited  by 
any  contained  acid  or  salt,  except  the  ammo  compounds.  In  addi- 
tion to  this  action  of  the  living  cells,  the  retarding  action  of  the  outer 
coat,  with  its  possible  differential  action  with  respect  to  the  acid,  salt, 
and  hydroxid,  are  to  be  taken  into  account.2 

1  Stiles,  Walter,  and  Ingvar  J0rgensen.     Studies  in  permeability.     The  swelling  of  plant  tissue 
in  water  and  its  relation  to  temperature  and  various  dissolved  substances.     Annals  of  Bot.,  31 : 
415.     1917.     See  also  Stiles  and  J0rgensen.     Quantitative  measurement  of  permeability.     Bot. 
Gazette,  65:526.     1918. 

2  Denny,  F.  E.     Permeability  of  certain  plant  membranes  to  water.     Bot.  Gazette,  63 : 373-397. 
1917.     Permeability  of  membranes  as  related  to  their  composition.     Bot.  Gazette,  63:  468- 
485.     1917. 


Water  Deficit  or  Unsatisfied  Hydration  Capacity,  101 

It  is  to  be  added  that  when  the  dried  slices  of  the  commercial  prep- 
aration of  potatoes  known  as  "Anhydrous"  were  tested  at  22°  to  23° 
C.,  the  sections,  which  were  1  to  1.1  mm.  in  thickness,  showed  a 
maximum  swelling  of  265  per  cent  in  potassium  hydroxid  0.01  M, 
250  per  cent  in  citric  acid  (0.01  N),  and  200  per  cent  in  acidified 
potassium  chloride,  at  0.01  N  concentration,  while  the  swelling  in  dis- 
tilled water  was  222  per  cent.  The  relatively  lessened  swelling  in 
distilled  water,  as  compared  with  salts  and  acids,  might  be  attributed  to 
the  elimination  of  the  osmotic  action  of  the  semi-permeable  membrane. 
The  exact  history  of  the  preparation  of  the  material  is  not  available, 
however,  and  here,  as  in  other  dried  material,  the  coagulatory  effects 
of  contained  salts  and  acids  in  desiccation  must  be  taken  into  account. 
This  will  become  apparent  from  the  results  obtained  from  the  swelling 
of  dried  apples  made  from  machine-cut  strips.  Sections  cut  from 
these  strips  had  an  average  thickness  of  2.6  to  3.4  mm.  and  gave 
swelling  increases  as  follows  at  16°  to  18°  C.: 

TABLE  80. 

p.  ct. 

Distilled  water 72 

Citric  acid,  0.01  N 69 

Sodium  hydroxid  0.01  M 105 

Calcium  chloride,  3  M 123 

The  expected  proportionate  swellings  in  water,  acids,  and  alkaline 
solutions  are  noted,  but  the  great  imbibition  from  the  solution  of 
calcium  chloride  which  would  afford  an  osmotic  pressure  of  68  atmos- 
pheres is  a  fact  of  extraordinary  interest.  An  extension  of  the  test 
was  made  in  different  concentrations  of  this  salt,  in  which  it  was  found 
that  at  19°  to  20°  C.,  swellings  of  82  and  95  per  cent  only  were  obtained 
in  a  solution  of  0.01  M  and  in  other  tests  of  the  original  concentrated 
solution;  3  M  gave  an  increase  of  230  per  cent  on  one  trial  and  106 
per  cent  on  another  at  the  above  temperatures.  This  maximum  was 
obtained  from  sections  1  mm.  in  thickness,  and  it  is  suggested  that 
they  were  in  a  state  of  undue  compression.  Similar  sections  swelled 
180  per  cent  in  a  2.7  M  solution  of  potassium  nitrate  capable  of  exert- 
ing an  osmotic  pull  of  84  atmospheres.1  Thicker  sections  in  the  potas- 
sium solutions  of  this  concentration  swelled  only  62  per  cent  and  gave 
an  expected  greater  swelling  of  94  per  cent  in  a  0.01  N  solution.  The 
maximum  swelling  in  the  concentrated  calcium  solution  suggests  that 
compounds  of  the  calcium  with  the  pectin  may  be  formed  of  higher 
hydration  value,  which  makes  possible  these  unexpected  imbibition 
reactions.  Such  swellings,  however,  have  been  found  hi  the  case  of 
colloidal  mixtures  which  simulate  the  action  of  the  plant  in  concen- 
trated solutions  of  potassium  nitrate. 

1  MacDougal  and  Spoehr.  The  behavior  of  certain  gels  useful  in  the  interpretation  of  the  ac- 
tion of  plants.  Science,  45:484-488.  1916. 


102 


Hydration  and  Growth. 


The  cell-sap  of  Echinocactus  grown  at  the  Desert  Laboratory  has  a 
low  content  of  solid  matter,  and  shows  osmotic  pressures  of  3  to  5 
atmospheres,  calculated  by  cryoscopic  methods.  The  acidity  of  the 
massive  body  is  greatest  in  the  external  layers  and  decreases  toward  the 
center,  and  also  shows  daily  variation  which  is  greatest  in  the  exter- 
nal layer.  This  is  illustrated  by  the  data  in  table  81,  obtained  by 
Mr.  E.  R.  Long,  in  which  A  designates  the  external  layer  and  D 
the  innermost  parenchyma,  B  and  C  being  intermediate.1 

TABLE  81. 


Plant. 

Weight. 

Dates  and 
hours. 

Sample. 

Afternoon. 

Morning. 

Dry  wt. 

Acidity. 

Dry  wt. 

Acidity. 

kg. 

No.  27. 

20 

June  30- 

A 

11.9 

0.376 

10.0 

0.342 

July  1.  .  . 

B 

10.7 

.269 

9.0 

.278 

4  p.  m., 

C 

7.7 

.154 

7.5 

.166 

8  a.  m.  .  . 

D 

6.7 

.143 

7.1 

.166 

The  freshly  expressed  juice  of  regions  C  and  D  of  such  a  plant  at 
midday  caused  sections  of  a  biocolloid  consisting  of  5  parts  agar,  3 
parts  mucilage  of  Opuntia,  and  1  each  of  gelatine  and  bean  protein  to 
swell  about  840  per  cent  at  20°  C.,  while  similar  sections  increased 
about  2,500  pe^  cent  in  distilled  water.  Dried  slices  of  the  paren- 
chymatous  tissue  similar  to  that  from  which  the  juice  was  expressed 
swelled  115  per  cent  in  the  juice,  while  they  increased  but  42  per  cent 
in  distilled  water  at  the  same  temperature. 

Some  of  the  sap  of  Echinocactus  caused  dried  slices  of  Opuntia,  the 
swelling  of  which  has  been  described  in  detail  in  Chapter  VII,  to  swell 
372  per  cent  at  20°  C.,  which  is  to  be  compared  with  550  per  cent  in 
distilled  water.  It  is  notable,  however,  that  such  dried  sections  of 
Opuntia  increased  325  per  cent  in  the  sap  expressed  from  living  joints 
of  the  same  kind,  thus  swelling  less  in  its  own  sap  than  in  that  of 
Echinocactus.  It  seems  probable  that  the  possibilities  of  parasitism 
might  be  more  profitably  sought  in  these  hydration  relations  rather 
than  in  the  simpler  osmotic  coefficients  to  which  the  author  attributed 
great  importance  in  his  original  study  of  this  matter.2 

It  is  highly  probable  that  any  cell-mass  would  absorb  water  and 
swell  in  fresh  sap  from  other  cell-masses  of  the  same  kind  in  an  equiva- 
lent condition.  Kunkel  placed  spores  of  Monilia  sitophila  (Mont)  in 
the  sap  of  equivalent  spores  and  obtained  no  plasmolytic  effects  and 
did  not  measure  for  swelling.  When  the  sap  was  reduced  to  one-tenth 

1  Long,  E.  R.     Acid  accumulation  and  destruction  in  large  succulents.     The  Plant  World,  18: 
No.  10,  261.     1918. 

2  MacDougal  and  Cannon.     The  conditions  of  parasitism    in  plants.     Carnegie  Inst.  Wash. 
Pub.  No.  129,  1910.     See  also  MacDougal,  The  beginnings  and  physical  basis  of  parasitism.    The 
Plant  World,  20:  p.  238.     1917. 


Water  Deficit  or  Unsatisfied  Hydration  Capacity.  103 

of  its  volume  by  boiling,  spores  immersed  in  the  concentrate  plasmo- 
lyzed,  as  might  be  expected.  The  temperature  implied  would  of  course 
cause  many  changes  in  the  constitution  of  the  sap.  The  fresh  juice 
was  seen  to  cause  plasmolysis  in  vegetative  cells  of  Spirogyra  setiformis.1 

In  any  consideration  of  the  general  facts  which  are  brought  into  a 
discussion  of  permeability  in  the  experimental  laboratory,  one  of  the 
most  disturbing  features  is  the  frequency  with  which  results  are  en- 
countered which  can  not  be  duplicated.  The  effects  of  phlorizin  on 
the  diffusion  of  sugar  is  an  example  of  such  a  matter.  Preparations 
of  the  fresh  and  turgid  inner  layers  of  onions  prepared  by  Dr.  H.  A. 
Spoehr  were  found  to  contain  an  amount  of  sugar  suitable  for  experi- 
mentation, and  when  such  sections  were  placed  in  distilled  water  for 
a  short  period  the  amount  of  sugar  extracted  was  fairly  equivalent 
to  that  coming  out  of  other  sections  placed  in  the  solution  of  potas- 
sium carbonate  and  phlorizin  (0.02  M)  customarily  employed  in  the 
experiments  with  this  glucoside,  and  also  to  the  amount  of  sugar 
which  was  found  to  diffuse  out  of  sections  placed  in  the  potassium- 
carbonate  solution  alone.  Wachter  believed  he  had  proved  that  the 
addition  of  a  trace  of  potassium  salt  to  water  would  prevent  the 
diffusion  of  sugar  from-  tissue  of  onions.2 

Sections  of  the  layers  of  onion  bulbs  were  placed  in  distilled  water 
and  in  solutions  of  phlorizin  and  potassium  carbonate,  to  ascertain 
to  what  relative  extent  they  might  influence  hydration.  Trios  of  sec- 
tions having  an  average  thickness  of  about  2.3  mm.  were  prepared 
for  measurement  under  the  auxograph  and  a  series  of  three  prepara- 
tions were  swelled  at  20°  C.  The  set  in  water  increased  1.5  per  cent, 
that  in  the  potassium  carbonate  4.3  per  cent,  and  the  one  in  potas- 
sium carbonate  and  phlorizin  9  per  cent,  the  maximum  amount. 

In  the  repetition  of  the  above  with  other  material  the  results  were 
somewhat  different.  It  was  found  that  trios  at  20°  C.  swelled  4  to  8 
per  cent  in  water,  7  to  11  per  cent  in  the  potassium-carbonate  solutions, 
and  between  5  and  6  per  cent  in  the  solution  of  phlorizin  and  potas- 
sium carbonate.  Such  results  are  indicative  of  an  inequality  of  the 
material,  but  it  is  evident  that  not  only  does  potassium  carbonate 
not  prevent  the  diffusion  of  sugar  from  the  onion,  but  that  a  hydra- 
tion of  greater  amount  may  occur  in  its  presence  than  in  water  alone. 
Nothing  could  be  determined  as  to  the  action  of  the  phlorizin  on  plant 
colloids  with  relation  to  sugar.3 

It  has  also  been  found  impossible  to  bring  the  results  of  theoretical 
antagonisms  of  substances  taken  up  by  protoplasm  into  harmony 
with  the  results  of  hydrations  made  in  the  present  connection,  a  matter 

1  Kunkel,  Louis  Otto.  A  study  of  the  problem  of  water  absorption.  23d  Ann.  Rep.  Missouri 
Botanical  Garden,  pp.  26-40.  1912. 

*  Wachter,  W.  Untersuchungen  ileber  den  Austritt  von  Zucker  aus  den  Zellen  der  Speicher- 
organe  von  Allium  cepa  und  Beta  vulgaris.  Jahrb.  f.  wiss.  Bot.,  41:  165.  1905. 

3  See  Brooks,  S.  C.     Permeability  of  cell-walls  of  Allium.     Bot.  Gaz.,  64:509.     1917. 


104  Hydration  and  Growth. 

which  for  the  present  may  be  ascribed  in  part  to  the  fact  that  generaliza- 
tions on  permeability  rest  upon  results  obtained  with  a  narrow  range 
of  material  or  under  highly  specialized  conditions.  It  would  seem 
allowable  to  assume,  if  antagonistic  effects  are  to  be  attributed  to 
the  opposed  action  of  two  salts,  that  sections  of  living  plants  would 
show  differentiating  swelling  capacities  in  balanced  solutions  and  in 
their  two  components.  The  first  trial  of  this  matter  was  made  with 
terminal  internodes  of  Mentha  spicata,  which  had  attained  about  half 
of  their  final  length  and  had  an  average  diameter  of  3  mm.  The 
imbibition  capacity  of  this  plant  in  living  and  dried  conditions  has 
been  described  elsewhere  in  this  paper.  An  estimation  by  Professor 
H.  M.  Richards  made  the  approximate  acidity  of  the  "pure  juice" 
such  that  1  c.c.  =  0.45  c.c.  N/20  KOH,  and  that  the  total  acidity  of  a 
gram  of  fresh  material  was  equivalent  to  0.64  c.c.  N/20  KOH. 

A  few  simple  tests  were  planned  which  might  furnish  results  having 
a  bearing  upon  the  present  question.  The  neutralizing  or  balancing 
action  of  sodium  and  calcium  being  one  of  the  commonest  conjunctions, 
the  action  of  these  two  elements  in  the  form  of  chlorides  were  tried.1 

The  balanced  solution,  consisting  of  100  parts  of  0.375  M  sodium 
chloride  and  10  parts  0.195  M  calcium  chloride  was  used.  Such  a 
solution  plasmolyzes  cells  of  Spirogyra,  but  according  to  Osterhout's 
findings,  the  presence  of  two  salts  in  the  proportions  given  prevents 
either  from  passing  the  membrane.  It  would  appear  that  Osterhout 
in  later  papers  takes  the  position  that  the  essential  feature  of  antagon- 
ism between  two  substances  consists  in  the  fact  that  they  produce 
opposed  effects  upon  it.2 

Sections  of  stems  of  Mentha,  as  described  above,  swelled  about 
0.075  mm.  in  distilled  water,  shrunk  1  mm.  in  the  balanced  solution, 
but  came  back  to  a  volume  slightly  greater  than  the  initial  size  when 
distilled  water  was  run  into  the  dish.  The  shrinkage  in  the  solution 
of  sodium  chloride,  0.375  M,  was  very  marked,  dilution  being  fol- 
lowed by  a  resumption  of  the  original  volume.  Similar  effects  were 
obtained  with  the  solution  calcium  chloride. 

The  terminal  parts  of  growing  stems  of  an  Erigeron,  which  were 
higher  in  acid  than  Mentha,  were  next  tested.  The  sections  had  a 
diameter  of  about  3.7  mm.  and  included  a  length  of  5  to  7  mm.  of  the 
stem.  The  balanced  solution  and  its  constituents  were  diluted  to 
one-thirtieth  of  the  above  concentration  to  avoid  shrinkage  by  plas- 
molysis.  The  swelling  in  distilled  water  (average  of  3  sections  by 
auxographic  method)  was  0.2  mm.;  in  the  balanced  solution,  0.15 
mm.;  in  sodium  chloride  (6  sections),  0.2  mm.;  and  in  calcium  chlo- 
ride (6  sections),  0.15  mm.  These  minute  swellings  progressed  for  a 

1  Osterhout,  W.  J.  V.     The  permeability  of  living  cells  to  salts  in  pure  and  balanced  solutions. 
Science,  34:  187-189.     1911. 

2  Osterhout,  W.  J.  V.     Nature  of  antagonism.     Science,  41 : 255-256.     1915. 


Water  Deficit  or  Unsatisfied  Hydration  Capacity. 


105 


period  of  9  or  10  hours  (inclusive  of  the  action  of  distilled  water). 
The  sections  then  slowly  shrunk  at  rates  which  would  carry  them  back 
to  original  volume  in  a  similar  period.  The  swelling  in  the  balanced 
solution  was  equivalent  to  the  effect  of  its  calcium,  while  the  sodium 
alone  gave  a  greater  swelling  than  the  balanced  solution.  The  swell- 
ing in  this  salt  was  as  great  as  in  distilled  water. 

Sections  of  young  joints  of  Opuntia  have  been  subjected  to  a  wide 
variety  of  tests,  and  a  set  of  these  was  placed  under  conditions  similar 
to  those  noted  for  Erigeron.  The  swelling  in  distilled  water  was  7.1 
per  cent,  dilute  balanced  solution  8.7  per  cent,  sodium  chloride  7.2 
and  8.2  per  cent,  and  in  calcium  chloride  7.7  and  8.8  per  cent.  The 
small  differences  in  the  proportionate  swelling  do  not  appear  to  have 
any  bearing  on  possible  antagonisms. 

The  masses  of  tissue  used  in  the  above  tests  were  complex  as  to 
composition  and  doubtless  already  contained  some  of  the  salts  of  the 
balanced  pair.  It  seemed  important  to  test  the  effect  of  antago- 
nistic salts  on  a  biocolloid  inclusive  of  bean  protein,  and  a  simpler  one 
in  which  agar  was  combined  with  glycocoll.  The  results  of  the  meas- 
urements were  as  shown  in  table  82. 

TABLE  82. 


Agar  90  parts, 
bean  protein  10  parts, 
0.23  mm.  in  thickness. 

Agar  90  parts, 
glycocoll  10  parts, 
15  mm.  in  thickness. 

Distilled  water  

p.  ct. 
569.6 
195.6 
195.6 
228.3 
195.6 

p.  ct. 

p.  ct. 
1,133  3 

p.  ct. 

Balanced  solution  

239.1 

466.6 
400 
533.3 
333.3 

533.3 

Calcium  chloride,  0.195  M.  . 
Sodium  chloride,  0.375  M.  .  . 
Sodium  hydroxid,  0.01  M  .  . 

The  amount  of  imbibition  hi  the  balanced  solution  in  both  colloids 
was  less  than  half  that  in  distilled  water,  and  was  not  much  different 
from  that  in  the  sodium  chloride.  The  amount  of  imbibition  hi  the 
calcium  component  used  separately  was  less  than  that  in  the  sodium 
chloride  alone.  No  aspect  of  the  above  experimentation  seems  to 
promise  anything  of  importance  concerning  the  nature  of  the  external 
layer  of  the  cell,  differential  action  of  which  to  solutions  of  various 
kinds  is  so  largely  a  matter  of  assumption.  In  fact,  but  little  of  the 
evidence  obtained  by  the  extended  experiments  described  in  this 
volume  requires  such  a  conception  for  its  explanation,  a  conclusion 
previously  set  forth  very  clearly  by  Kunkel  after  an  experimental 
examination  of  some  features  of  the  reactions  of  cells  supposedly 
associated  with  semi-permeable  membranes.1 

1  Kunkel,  Louis  Otto.     A  study  of   the  problem  of   water   absorption.     23d   Ann.  Report, 
Missouri  Botanical  Garden,  pp.  26-40.     1912. 


106  Hydration  and  Growth, 

The  swelling  of  dried  sections  is  clearly  under  conditions  exclusive 
of  the  action  of  a  semi-permeable  membrane,  except  in  so  far  as 
the  cell-walls  may  show  such  properties.  If  desiccation  resulted  in 
simple  loss  of  water  such  as  that  which  ensues  in  an  unsalted  plate 
of  biocolloids,  the  action  of  living  and  dried  material  might  be  expected 
to  be  identical.  The  presence  of  salts  and  acids,  however,  causes  some 
irreversible  changes,  and  the  relative  swelling  of  dried  sections  in  various 
solutions  is  different  from  that  of  a  series  obtained  from  the  use  of  liv- 
ing cell-masses.  That  the  cell  does  act  as  an  osmotic  machine  is  estab- 
lished beyond  all  question.  That  it  is  an  enormously  complex  system 
of  osmotic  sacs  is  well  established.  That  the  differential  action  of  the 
specialized  layers  formed  at  all  phase  boundaries  can  be  made  to 
account  for  the  entire  relations  of  the  protoplast  is  more  than  doubtful. 
While  all  hydration  in  the  broadest  sense,  including  osmotic  action, 
ultimately  depends  upon  molecular  affinity,  it  is  evident  that  the  con- 
ception of  the  semi-permeable  sac  does  not  offer  a  suite  of  possibilities 
which  may  account  for  the  range  of  action  of  the  protoplast. 

A  second  proposal  is  that  which  has  been  most  clearly  outlined  by 
Dr.  E.  E.  Free,  based  upon  the  general  acceptance  of  specialization  of 
colloidal  conditions  in  the  external  layer  of  the  protoplast,  but  explains 
its  action  upon  changes  in  the  relations  of  the  colloidal  phases  in  it.1 

Shrinkage  of  such  a  cell  would  follow  a  change  in  the  dispersion  or 
phase  relations  of  the  external  layer,  and  might  be  accompanied  by  the 
solvation  or  passage  out  of  the  cell  of  substances  which  would  reduce 
its  water-holding  capacity  still  further.  The  explanation  based  on 
osmosis  might  account  for  the  escape  of  the  free  liquid  of  the  vacuoles, 
or  from  syncretic  cavities,  but  some  mechanism  such  as  that  suggested 
by  Free  is  necessary  to  account  for  the  lessening  of  the  amount  of 
water  held  by  the  cell  colloids. 

Some  further  evidence  on  the  matter  of  extraction  of  substances 
from  colloidal  masses  and  the  action  of  contained  substances  on  desic- 
cating colloids  remains  to  be  considered.  The  facts  which  seem  to 
warrant  the  prolongation  of  the  discussion  were  obtained  by  a  com- 
parison of  the  results  of  swelling  of  living  and  dried  sections,  with 
determinations  of  their  acidity,  measurements  of  substances  extracted, 
and  measurements  of  repeated  swellings  of  the  same  material. 

These  treatments  as  applied  to  median  slices  of  maturing  joints  of 
Opuntia  discata  grown  at  Carmel  gave  measurements  at  18°  C.  as 
shown  in  table  83. 

The  second  swelling  of  dried  sections  which  had  been  once  swelled 
(extracted)  indicate  that  some  material  of  high  hydration  capacity  had 
been  dissolved  out,  as  the  sections  which  had  been  simply  dried  swelled 
25  times  as  much  in  potassium  nitrate.  Material  which  was  dried 
directly  without  extraction  did  not  undergo  any  lessened  hydration 

1Free,  E.  E.   A  colloidal  hypothesis  of  protoplasmic  permeability.    Plant  World,  21:141.    1918. 


Water  Deficit  or  Unsatisfied  Hydration  Capacity. 


107 


capacity  by  swelling,  as  it  swelled  a  second  time  to  the  same  amplitude, 
except  in  the  acid  solution. 


TABLE  83. 


Opuntia  discata. 

Living 
median 
slices. 

Same  sections  dried. 
Swelling  calculated 
on  basis  of 
original  thickness. 

Dried  median  slices  not 
previously  treated. 

First  swelling. 

Second  swelling 
after  drying. 

Distilled  water  

p.  ct. 
11.4 
6.4 
6.6 
9.2 

p.  ct. 
17.5 
26.4 
24.5 
22.4 

p.  ct. 
430 
357 
315 
541 

p.  ct. 
430 
250 
352 
541 

Citric  acid,  0.01  N  

Potassium  hydroxid,  0.01  M. 
Potassium  nitrate,  0.01  M  .  . 

TABLE  84. 


Opuntia  discata, 
median  slices. 

First 
swelling. 

Second 
swelling. 

Distilled  water  

p.  ct. 
225 

p.  ct. 
100 

Citric  acid,  0.01  N  

225 

107 

Sodium  hydroxid,  0.01  M  .  .  . 
Potassium  hydroxid,  0.01  M  . 

281 

287.5 

87.5 
137.5 

TABLE  85. 


Swelling  of  fresh  material  implies  bursting  of  cells  by  combined 
imbibition  and  absorption  and  the  consequent  escape  of  the  mucilages 
which  would  not  occur  hi  the  hydration  of  dried  sections,  and  these 
would  consequently,  in  reswelling,  regain  their  original  dimensions,  or 
repeat  the  first  swelling.1 

That  this  action  depends 
on  the  condition  of  the  cell- 
masses  was  demonstrated  by 
the  fact  that  another  series 
of  sections  of  the  same  plant 
which  included  the  chloro- 
phyllose  layer  and  the  epi- 
dermis did  not  show  such  du- 
plication of  results  on  the 
second  swelling.  The  in- 
creases at  16°  to  18°  C.  were 
as  shown  in  table  84. 

It  would  be  unsafe  to  as- 
sume that  such  a  result  is 
associated  only  with  a  chloro- 
phyll-bearing tissue,as  median 
slices  of  a  second  opuntia 
with  a  smaller  proportion  of 
mucilage  gave  similar  swell- 
ings at  16°  C.,  as  shown  in  table  85. 

The  lessened  swelling  in  this  case  can  not  be  attributed  to  the  escape 
of  pentosans  from  bursting  cells,  and  attention  naturally  is  directed 
to  the  readily  diffusible  amino-acids,  the  presence  of  which  facilitates 
hydration  in  a  remarkable  way. 

The  swelling  of  the  pentosan  agar,  which  has  been  used  so  widely 
in  the  imbibition  measurements  in  connection  with  growth,  would  be 

1  MacDougal  and  Spoehr.  The  solution  and  fixation  accompanying  swelling  and  drying  of 
biocolloids  and  plant  tissues.  Plant  World,  22:  June  1919. 


Opuntia  discata, 
median  slices. 

After 
first 
drying. 

After 
second 
drying. 

p.  ct. 

p.  ct. 

Distilled  water  

361 

42 

Citric  acid,  0.01  N  

306 

56 

Potassium  hydroxid,  0.01  M  . 

250 

100 

Potassium  nitrate,  0.01  M  .  . 

325 

75 

108  Hydration  and  Growth. 

accompanied  by  the  solution  or  dispersion  of  material  from  the  exter- 
nal part  of  the  sections  and  by  the  diffusion  of  whatever  salts  or  acids 
might  be  present  in  the  interior  of  the  mass.  Sections  0.25  mm.  in 
thickness  swelled  2,420  per  cent  in  water,  and  material  equivalent  to 
200  similar  sections,  weighing  1.6731  g.,  placed  in  water  for  the  same 
length  of  time,  lost  0.2570  g.,  or  15  per  cent  of  the  total.  A  similar 
test  of  sections  composed  of  8  parts  agar  and  2  parts  gelatine  showed  a 
swelling  of  1,684  per  cent,  with  a  loss  of  18  per  cent  of  the  original 
weight  by  dispersion  or  solution  in  the  water. 

Sections  of  Opuntia  swelled  in  water  for  24  hours  at  16°  to  18°  C. 
lost  7  per  cent  of  the  average  total  dry  weight  of  similar  sections.  As 
much  of  the  dry  weight  is  insoluble  cell-wall,  it  is  to  be  seen  that 
the  actual  percentage  of  soluble  or  diffusible  material  extracted  was 
large,  a  fact  which  would  readily  account  for  the  lessened  reswelling  of 
sections. 

A  single  effort  was  made  to  ascertain  to  what  extent  the  acids  are 
extracted  in  the  hydration  of  living  cell-masses  of  Opuntia  and  in  dried 
sections  of  the  same.  Determinations  by  Professor  H.  M.  Richards  of 
the  acidity  of  the  water  in  which  fresh  slices  of  the  Opuntia  were  swelled 
showed  that  this  might  be  expressed  as  follows:  10  c.  c.  solution  from 
dish  in  which  set  of  fresh  sections  were  swelled  in  water  =  0.44  N/20 
KOH. 

Dried  slices  of  the  above  material,  when  swelled  in  water  24  hours, 
gave  a  solution  the  acidity  of  which  might  be  expressed  as  10  c.  c.  of 
solution  =  0.10  N/20  KOH. 

When  such  sections  were  immersed  in  citric  acid  0.01  N,  the  strength 
of  the  solution  was  increased  so  that  at  the  end  of  24  hours  the  acidity 
was  expressible  as  10  c.  c.  of  solution  =  2.25  N/20  KOH.1 

The  extraction  of  acid  from  the  fresh  sections  in  water  is  marked 
and  is  much  greater  in  the  acid  solution.  This  action  in  setting 
free  the  amino-acids  would  cause  a  loss  in  hydration  capacity  which 
would  become  apparent  on  reswelling. 

The  chief  interest  in  the  present  work  is  centered  in  the  hydration 
of  protoplasm  associated  with  growth  The  development  of  embryonic 
cells  from  the  stage  of  a  highly  granular,  dense  colloidal  mass  with  a 
large  nucleus  to  maturity  is  characterized  by  the  migration  of  pro- 
teinaceous  material  from  the  nucleus  into  the  remainder  of  the  mass; 
by  the  formation  of  syncretic  cavities,  including  those  designated  as 
vacuoles;  and  by  a  constantly  varying  metabolism  which  results  in 
continuous  alterations  in  the  composition  of  the  vacuolar  fluids,  and 
in  the  composition  and  hydration  capacity  of  the  protoplasm.2 

The  peripheral  part  of  the  colloidal  mass  is  probably  of  greater 
density  than  the  interior,  and  it  is  the  behavior  of  this  layer  which 

1  MacDougal,  Richards,  and  Spoehr.     The  basis  of  succulence  in  plants.     Bot.  Gaz.,  67:405. 
1919. 

2  See  Thoday,  D.    On  turgescence  and  the  absorption  of  water  by  the  cells  of  plants.    The 
New  Phytologist,  17: 108.     1918. 


Water  Deficit  or  Unsatisfied  Hydration  Capacity.  109 

gives  rise  to  most  of  the  phenomena  known  as  permeability  and  osmosis. 
The  external  layer,  as  well  as  the  entire  colloidal  mass,  reacts  to  solu- 
tions in  a  manner  determined  by  its  composition  and  its  history, 
especially  with  respect  to  the  salts  which  have  come  into  the  cell  since 
its  formation,  and  particularly  to  the  salts  which  may  be  dissolved  in 
the  water  in  which  the  cell  is  immersed.  The  absorption  of  such  salts 
alters  the  capacity  of  the  colloids  in  various  ways.  In  the  case  of  the 
alkaline  solutions,  it  has  already  been  pointed  out  that  nearly  all  plant 
tissues  show  a  long-continued  swelling  which  may  be  reasonably 
ascribed  to  the  formation  of  a  salt  between  the  sugar  and  the  hydroxide, 
which  salt  has  a  greater  capacity  for  hydration  than  the  carbohy- 
drates alone. 

Acids  decrease  the  hydration  capacity  of  agar  and  of  the  pentosans 
which  enter  into  the  composition  of  certain  types  of  plant  cells,  of 
which  the  tissues  of  the  potato  would  be  a  good  example. 

The  death  of  cell-masses  by  desiccation  probably  produces  less  dis- 
arrangement of  the  material  of  the  cell  than  any  other  method  of 
killing,  and  not  only  the  colloidal  mass,  but  its  external  layer,  probably 
retains  its  condition  with  respect  to  phases  with  but  little  serious 
disturbance.  The  only  positive  movement  which  might  be  recog- 
nized as  of  importance  in  connection  with  the  point  now  being 
discussed  would  be  that  as  drying-out  proceeds  the  salts  and  acids 
of  the  mass  would  pass  toward  the  periphery  and  might  accumulate 
at  that  place,  although  it  could  by  no  means  be  assumed  that  such 
action  would  result  in  a  uniform  deposition  in  the  membrane.  The 
hydration  of  such  dried  material  would  take  place  as  in  other  dead 
material,  being  largely  by  means  of  imbibition  and  adsorption,  with 
osmosis  by  the  action  of  vacuolar  material  almost  eliminated.  The 
records  of  many  measurements  of  dried  plants,  of  which  fresh  and 
living  portions  have  been  tested,  as  described  in  this  chapter,  are 
meant  to  be  explanatory  and  illustrative,  but  they  show  conclusively 
that  any  serious  development  of  our  conceptions  of  the  water-rela- 
tions of  cell-masses  must  be  based  upon  and  take  seriously  into  con- 
sideration the  hydration  processes  of  the  protoplastic  colloids. 

Colloids  are  never  at  rest,  yet  it  is  possible  to  secure  combinations 
of  conditions  in  mixtures  in  which  hydration,  for  example,  is  all  but 
complete.  Protoplasm,  however,  is  the  seat  of  complex  transforma- 
tions and  is  the  medium  of  such  diverse  diffusion  movements  that 
ideally  it  is  never  in  a  condition  of  satisfied  hydration.  The  amounts 
which  it  may  take  up  from  different  solutions  and  under  various  con- 
ditions previously  described  may,  on  proper  analysis,  serve  to  show 
the  nature  of  the  protoplastic  colloid  with  respect  to  its  principal 
components.  The  determination  of  the  index  of  unsatisfied  hydra- 
tion, or  the  water  deficit,  is  an  indispensable  feature  of  any  serious 
effort  to  analyze  growth  into  its  physical  components. 


IX.  TEMPERATURE  AND  THE  HYDRATION  AND  GROWTH 
OF  COLLOIDS  AND  OF  CELL-MASSES. 

Living  material  is  a  colloidal  mass  consisting  of  a  mixture  of 
colloids,  the  most  important  components  of  which  are  carbohydrates 
and  proteins  or  protein  derivatives.  Salts  of  sodium,  potassium,  and 
magnesium  in  various  combinations  are  dissolved  in  the  system.  The 
denser  portions  of  the  protoplasm,  including  all  of  the  continuing 
structures,  or  those  of  morphological  rank,  have  the  properties  of  an 
elastic  gel  with  the  general  structure  of  a  fine  sponge  or  a  honeycomb 
with  irregularly  broken  or  incomplete  walls.  The  materials  which 
make  up  this  fairly  continuous  structure  are  also  in  a  disperse  or  liquid 
condition  in  the  cavities  and  may  even  fill  large  syncretic  spaces  in  the 
general  structure. 

The  essential  feature  of  growth  consists  in  the  accretion  of  material 
entering  into  this  colloidal  structure,  its  hydration,  and  its  arrangement 
into  additional  structures  or  portions  of  honeycomb.  This  may  be 
pictured  as  taking  place  by  an  initial  increased  dispersion  or  enlarge- 
ment of  the  colloidal  network  to  a  point  where  new  masses  of  gel  would 
be  formed  in  the  liquid  phase  of  the  existing  mesh.  Considering  living 
material  as  an  intimate  mixture  of  minute  particles  of  its  main  colloidal 
components  (and  the  scanty  evidence  on  this  matter  is  to  the  effect 
that  the  carbohydrates  and  proteins  do  not  diffuse  into  each  other),  it 
is  on  this  basis  to  be  assumed  that  the  new  material  would  accrue  to 
these  separately  and  in  a  characteristic  and  differentiated  manner. 

Aggregations  of  the  introduced  material  might  also  take  place  in 
syncretic  cavities,  with  opportunity  for  the  development  of  specialized 
structures.  The  absorption  and  diffusion  of  material  in  liquid  form 
and  its  diffusion  into  the  colloidal  mass  would  in  all  cases  be  the  initial 
step  in  growth.  The  consequent  swelling  with  all  of  its  accompani- 
ments and  consequences  in  cell-masses  of  plants  and  in  my  biocolloidal 
mixtures  has  been  found  to  depend  upon  the  character  and  the  pro- 
portion of  the  proteins  or  protein  derivatives  in  the  colloid,  the  pro- 
portion of  the  pentosans,  and  the  amount  of  salts  present.  In  addition 
to  these  features,  which  change  but  slowly,  the  active  metabolism  of 
the  growing  cell  includes  respiration  in  which  substances  such  as  sugars 
may  be  adsorbed  on  surfaces  of  aggregates  of  enzymes  which  catalyze 
them,  acids  occurring  at  certain  stages  of  the  resultant  reactions. 
Acidity  in  a  growing  cactus,  for  example,  may  vary  between  a  value  of 
0.1  N  malic  acid  and  one-twentieth  of  this  amount  during  the  course 
of  a  daylight  period,  causing  very  marked  changes  in  the  imbibition 
and  absorption  of  the  cell-colloids. 

The  exposure  of  a  growing  or  swelling  colloid  to  different  tempera- 
tures has  several  effects:  First,  the  rate  of  absorption  and  diffusion  of 

110 


Hydralion  and  Growth  of  Colloids  and  Cell-masses. 


Ill 


water  is  modified,  being  generally  accelerated  by  a  rise  within  the  range 
of  ordinary  temperatures  at  which  plants  grow.  Next,  adsorption  is 
affected  in  a  contrary  manner,  but  the  complex  series  of  reactions 
associated  with  respiration  are  speeded  up,  with  consequences  far  too 
complex  to  be  characterized  here.  The  first  step,  that  of  absorption 
and  diffusion,  would  be  far  simpler,  as  the  rate  of  acceleration  here 
would  be  nearly  identical  with  that  of  the  diffusion  of  the  same  material 
in  water. 

A  determination  of  the  effect  which  temperature  might  have  on 
growth  necessarily  takes  into  account,  first,  the  fundamental  increase 
which  might  accrue  from  simple  absorption  under  various  conditions. 
In  recognition  of  this  fact,  it  was  arranged  to  carry  on  swellings  of 
some  of  the  biocolloids  in  order  to  gain  some  appreciation  of  the  im- 
bibition factor  in  growth.  Sections  0.2  mm.  in  thickness  of  plates  of 
agar  90  parts,  bean  protein  10  parts,  and  culture  salts  0.85  per  cent 
were  swelled  at  15°  C.  and  at  22°  C.  The  rate  of  swelling  may  be 
appreciated  by  a  consideration  of  the  total  amounts  at  the  end  of  4 
hours,  8  hours,  and  at  the  end  of  the  test  (fig.  19). 


A  p.m. 


8p.m 


12p.m. A  a.m. 


8a.m. 


1 


T 


.  I5°C. 


22°C. 


30°C. 


39'C. 


[\ 


FIG.  19. — Tracing  of  auxographic  record  of  swelling  of  dried  plates  0.2  mm.  in  thickness  of  a 
salted  "biocolloid"  consisting  of  9  parts  of  agar,  1  part  bean  protein,  and  0.015  part 
nutritive  salts.  Increase  is  denoted  by  downward  movement  of  the  pen,  amplified  20  times. 
The  initial  rate  and  continuance  of  the  swelling  at  various  temperatures  may  be  compared. 

TABLE  86. — Swelling  of  mixture  of  agar,  bean  protein,  and  cultures  in  distilled  water. 


Swellings,  per  cent. 

Averages. 

4  hours  (15°  C.)  . 

800 

875 

838 

8  hours  (15°  C.)  . 

1,150 

1,075 

1,113 

22  hours  (15°  C.)  . 

1,325 

1,325 

1,325 

4  hours  (22°  C.)  . 

1,325 

1,450 

1,388 

8  hours  (22°  C.)  . 

1,400 

1,700 

1,550 

22  hours  (22°  C.)  . 

1,900 

1,900 

1,900 

The  hydration  during  the  first  4  hours  includes  any  process  of 
chemical  union  of  water  with  the  colloidal  material  by  which  water 
in  definite  proportions  enters  into  the  molecular  aggregates.  The 
greater  part  of  this  action  probably  takes  place  within  the  first  half 


112 


Hydration  and  Growth. 


hour  of  immersion,  with  consequent  great  rapidity  of  swelling.  It  is 
not  possible  to  separate  this  action  from  adsorption,  which  follows  more 
slowly,  but  if  the  first  4-hour  period  be  considered,  it  is  to  be  seen  that 
the  total  rises  from  about  9  to  14,  with  a  change  from  15°  to  22°  C.  at 
which  the  immersion  was  made.  If  the  first  8  hours  is  considered,  the 
hydration  as  expressed  by  the  total  swelling  is  as  11  to  16  at  the  two 
temperatures  and  as  13  to  19  for  the  entire  period  of  hydration,  a  ratio 
which  includes  the  accelerating  effects  of  a  rise  in  temperature  both  on 
the  initial  chemical  action  and  the  continued  absorption.1 

A  biocolloid  without  salts  consisting  of  9  parts  agar  and  1  part  oat 
protein  was  now  tested  for  comparison,  with  results  shown  in  table  87. 

TABLE  87. 


Swellings,  per  cent. 

Averages. 

4  hours  (15°  C.)  .  .      . 

1,111         1,222 

1,167 

8  hours  (15°  C.)  .  .      . 

1,417        1,500 

1,459 

23  hours,  final(15°C.     . 

1,722         1,866 

1,794 

4  houra  (23°  C.)  .  .      . 

1,278        1,333 

1,306 

8  hours  (23°  C.)  .  .      . 

1,944        1,861 

1,903 

23  hours  (23°  C.)  .  .      . 

2,528        2,444 

2,486 

The  swelling  during  the  initial  period  of  this  salt-free  mixture  is 
actually  greater  than  that  of  the  first  mixture,  but  is  affected  in  far 
less  degree  by  the  rise  in  temperature,  the  ratio  at  15°  C.  and  at  23°  C. 
being  as  12  to  13.  The  difference,  if  the  8-hour  period  is  considered, 


FIG.  20. — Tracing  of  auxographic  records  of  swelling  of  plates  0.18  mm.  in  thickness,  consisting 
of  9  parts  agar  and  1  part  oat  protein,  a  "  biocolloid "  with  very  high  hydration  capacity. 
The  5  mm.  and  4-hour  intervals  of  the  record  sheet  are  shown.  Increase  is  denoted  by  the 
downward  movement  of  the  pen,  which  amplifies  the  actual  swelling  20  times.  The  varia- 
tion in  initial  and  following  rates  and  time  necessary  for  hydration  are  shown. 

was  as  16  to  10,  and  for  the  23-hour  period,  as  18  to  25.  It  is  suggested 
that  the  chemical  action  during  the  beginning  stages  of  swelling  is  far 
less  important  than  in  the  salted  mixture  and  that  the  entire  set  of 
reactions  is  one  in  which  absorption  and  hydrolysis  play  the  determin- 

1  MacDougal.     The  relation  of  growth  and  swelling  of  plants  and  biocolloids  to  temperature. 
Proc.  Soc.  Exper.  Biol.  and  Med.,  15:48.     1917. 


Hydration  and  Growth  of  Colloids  and  Cell-masses. 


113 


ing  r61e.    A  much  more  pronounced  effect,  however,  was  secured  at  the 
temperature  of  31°  C.  (fig.  20). 

Plates  of  agar  90  and  oat  protein  10  parts  which  were  0.16  mm.  in 
thickness  were  swelled  at  a  temperature  of  31°  C.  at  intervals  of  2 
hours  or  more,  with  the  results  given  in  table  88. 


TABLE  88. 


Swellings,  per  cent. 

Averages. 

4  hours  

2,031 
2,556 
2,906 

2,312 
2,968 
3,250 

2,172 
2,763 
3,078 

8  hours  

20  hours  

The  acceleration,  as  expressed  by  the  total  during  the  first  4  hours, 
was  as  13  to  22  in  the  rise  from  23°  C.  to  31°  C.,  as  19  to  28  when  the 
first  8  hours  is  considered,  and  as  25  to  38  when  the  entire  swelling  is 
taken  into  account.  Raising  the  temperature  to  39°  C.  gave  swellings 
as  shown  in  table  89. 


TABLE  89. 


TABLE  90. 


Agar  90,  oat  protein  10. 

4  hours  

Swellings. 
p.  ct.           p.  ct. 
2,361          2,722 
2,555         3,166 
2,611          3,222 

Average, 
p.  ct. 
2,542 
2,861 
2,917 

8  hours  

12  hours  

Agar  90,  oat  protein  10. 

Swellings. 

p.  ct. 

4  hours  

2,555 

8  hours  

2,833 

10  hours  

2,888 

The  acceleration  accompanying  this  rise  of  8°  C.  was  followed  by 
an  increase  which  was  as  22  to  25  during  the  first  4  hours,  28  to  29 
during  the  second  8  hours,  and  the  hydration  for  the  entire  period  was 
actually  less  at  this  higher  temperature.  That  the  hydration  passed 
beyond  the  limits  of  acceleration  and  of  total  water-holding  capacity 
in  this  temperature  region  was  denoted  by  the  fact  that  swellings  at 
46°  to  47°  C.  were  as  given  in  table  90. 

TABLE  91. 


Swellings. 

Averages. 

4  hours  

1,444 
1,666 
2,083 

1,500 
1,805 
1,972 

1,472 
1,735 
2,022 

8  hours  

20  hours  

It  is  to  be  seen  that  while  the  final  capacity  for  swelling  at  15°  C. 
had  not  been  reached  in  20  to  22  hours,  it  was  practically  complete 
at  39°  C.  in  12  hours,  as  the  continuance  of  the  measurement  would 


114 


Hydration  and  Growth. 


have  detected  a  very  small  expansion,  while  satisfaction  was  nearly 
complete  in  8  hours  at  46°  to  47°  C. 

We  may  now  profitably  turn  to  an  extension  of  the  reactions  of  the 
salted  colloids  (see  p.  111).  Sections  of  agar,  bean  protein,  and  nutrient 
salts  0.18  mm.  in  thickness  gave  the  expansions  shown  in  table  91  at 
31°  C. 

The  temperature  series  was  taken  another  step  by  raising  the  air 
in  the  chamber  to  42°  C.,  at  which  point  the  liquid  in  the  dishes  showed 
38°  to  39°  C.,  being  cooled  by  evaporation  to  this  point.  The  measure- 
ments are  given  in  table  92. 

TABLE  92. 


Agar  90,  bean  protein  10,  nutrients  salts  0.85. 

4  hours  

Swellings 
p.  ct.           p.  ct. 
2,381         2,611 
2,638         2,833 
2,693         2,888 

Averages, 
p.  ct. 
2,486 
2,736 
2,791 

8  hours  

12  hours  

The  increase  of  the  temperature  of  the  air  in  the  chamber  to  52°  C. 
gave  a  temperature  of  46°  to  47°  C.  to  water  in  the  dishes.  The  swell- 
ings at  46°  to  47°  C.  are  shown  in  table  93. 


TABLE  93. 


Agar  90,  bean  protein  10,  nutrient  salts  0.85. 

4  hours  

Swellings, 
p.  ct.           p.  ct. 
2,000          2,305 
2,194          2,500 
1,111          2,500 

Averages, 
p.  ct. 
2,153 
2,347 
2,361 

8  hours  

10  hours  

A  review  of  the  action  of  the  salted  mixture  shows  a  very  slight 
acceleration  by  the  rise  in  temperature  from  22°  C.  to  31°  C.  and  also 
a  total  but  slightly  increased  above  that  of  the  lower  temperature. 
The  next  step,  from  31°  to  39°  C.,  however,  was  one  marked  by  a 
distinct  acceleration,  the  rates  during  the  first  4  hours  being  as  15  to 
25,  during  the  first  8  hours  as  17  to  27,  and  the  final  total  at  the  lower 
temperature  at  20  hours  was  as  20  to  28  at  the  higher  temperature  at 
the  end  of  12  hours. 

The  absorption  of  water  fell  off  at  temperatures  above  39°  C.,  which 
may  be  regarded  as  in  the  region  of  the  optimum  or  maximum  water- 
holding  capacity,  which  is  somewhat  lower  than  that  of  the  salt-free 
mixture.  Many  repetitions  of  the  tests  would  be  necessary  before  the 
matter  of  the  failure  to  show  expected  increase  of  hydration  between 
23°  and  31°  C.  was  accepted  as  a  reality.  The  measurements  in  ques- 


Hydration  and  Growth  of  Colloids  and  Cell-masses. 


115 


tion,  however,  suggest  that  even  in  hydrating  colloids,  in  which  meta- 
bolism is  not  in  progress,  abrupt  modifications  may  occur  by  the  con- 
junction or  disjunction  of  two  important  reactions  of  the  constellation 
present. 

The  chamber  in  which  these  tests  were  made  was  under  ground, 
with  earthen  and  board  walls,  and  was  reached  through  an  entrance 
chamber.  Electric  heaters  were  used.  The  chamber  was  2.7  by  2 
by  2.2  meters  and  it  was  necessary  to  enter  and  work  in  it  when  making 
the  tests.  The  air  was  stirred  by  a  fan  and  a  mercurial  thermometer 
suspended  from  the  ceiling  showed  air  temperatures  several  degrees 
above  that  of  the  water  in  the  dishes.  Thus,  in  the  preceding  test, 
the  observer  was  in  a  humid  atmosphere  at  55°  C.  (131°  F.)  when 
the  readings  of  the  swellings  were  48°  to  49°  C. 

The  compilation  of  averages  (table  94)  affords  a  ready  means  of 
comprehension  of  the  essential  features  of  the  entire  series. 

TABLE  94. 


4  hours. 

8  hours. 

20  to  22  hours. 

Agar  90,  bean  protein  10, 

culture  salts  0.85  : 

p.  ct. 

p.  ct. 

p.  ct. 

15  to  16°  C,  0.20  mm. 

888 

1,113 

1,325 

21  to  23°  C,  0.20  mm.  .  . 

1,388 

1,550 

1,900 

30  to  31°  C.,  0.18mm. 

1,472 

1,735 

2,022 

39°  C.,  0.18mm  

2,486 

2,736 

2,791  (12  hours). 

46  to  37°  C.,  0.18  mm.. 

2,163 

2,347 

2,361  (10  hours). 

48  to  49°  C.,  0.18  mm.. 

2,361 

2,514 

No  swelling  after  8 

hours. 

Agar  90,  oat  protein  10: 

15  to  16°  C.,  0.18  mm. 

1,167 

1,459 

1,794 

22  to  23°  C.,  0.16mm. 

1,388 

1,550 

1,900 

30  to  31°  C.,  0.16  mm. 

2,172 

2,763 

3,078 

38  to  39°  C.,  0.18  mm. 

2,541 

2,861 

2,917  (12  hours). 

46  to  47°  C.,  0.18  mm. 

2,555 

2,833 

2,889  (10  hours). 

48  to  49°  C.,  0.16  mm. 

1,906 

2,031 

No  swelling  after  8 

hours. 

A  series  of  swellings  of  agar  sections  0.18mm.  in  thickness,  made 
at  the  same  time,  affords  valuable  data  for  comparison  with  the  in- 
creases of  the  complex  biocolloids  (table  95). 

The  hydration  reactions  of  the  agar  are  not  so  positive  and  uniform 
as  those  of  the  more  complete  systems.  Increase  by  absorption  had 
not  reached  a  positive  final  in  24  hours  at  the  lowest  temperature.  A 
similar  stage  of  satisfaction  was  evident  in  half  this  time  at  about 
40°  C.  and  in  8  hours  at  49°  C.  The  rate  of  swelling  is  graphically 
illustrated  in  figure  21. 

The  maximum  or  " optimum"  of  swelling  of  such  agar  plates  occurs 
at  some  temperature  near  40°  C.  Initial  rate  and  total  increase  are 
greatest  at  this  point.  The  maximum  swelling  of  the  agar,  bean  pro- 


116 


Hydration  and  Growth. 


tein,  and  salt  mixture  lies  below  46°  C.  and  is  probably  above  40°  C., 
as  both  rate  and  total  capacity  become  uncertain  at  46°  C.  and  above. 

The  agar-oat  protein  mixture  has  a  higher  initial  capacity  at  46° 
to  47°  C.,  but  does  not  appear  to  absorb  as  much  water  as  it  did  at 


TABLE  95. 


Swelling  of  agar. 

4  hours. 

8  hours. 

10  hours. 

12  hours. 

16  hours. 

20  hours. 

24  hours. 

18  to  21°  C     . 

875 
806 
944 
1,000 
1,056 
1,292 
1,472 
1,750 
1,611 
1,306 
1,333 

961 
889 
1,083 
1,083 
1,167 
1,389 
1,556 
1,778 
1,667 
1,389 
1,417 

1,069 
1,000 
1,139 
1,278 
1,208 
1,417 
1,583 
1,833 

1,111 
1,054 
1,168 
1,333 
1,250 
1,444 

1,153 
1,083 
1,222 

27°  C  

29  to  31°  C  

39  to  41°  C  

40  to  41°  C  

1,708 

46  to  47°  C  

1,416 

48  to  49°  C  

Averages. 
18  to  21°  C     ... 

875 
1,028 
1,611 
1,320 

978 
1,125 
1,667 
1,403 

1,069 
1,244 
1,708 

1,111 
1,222 

1,153 

27°  C  

39  to  41°  C  

46  to  49°  C  

1,416 

a  temperature  a  few  degrees  lower.  The  probable  error  at  the  high- 
est temperatures  is  great,  however,  and  conclusions  as  to  a  separation 
of  initial  rate  and  final  capacity  should  be  made  with  caution.1 

The  relation  of  temperature  to  swelling  of  agar  and  of  these  colloidal 
mixtures  is  of  interest  because  of  the  fact  that  they  lie  within  the  range 
of  activities  of  plants.  The  greater  number  of  seed-forming  plants 


6p.m. 


12  p.m. 


8a.m. 


FIG.  21. — Tracing  of  auxographic  records  showing  swelling  of  plates  of  agar  0.18  mm.  in  thickness 
at  various  temperatures.  The  5  mm.  and  4-hour  intervals  of  the  record-slip  are  shown. 
Increase  is  denoted  by  the  downward  movement  of  the  pen,  which  amplifies  the  actual 
increase  20  times.  The  initial  and  following  rates  are  well  illustrated. 

do  not  endure  air-temperatures  of  above  45°  or  46°  C.  The  tempera- 
ture of  the  stems  or  growing-zones  in  such  exposures  can  not  be  cal- 
culated from  such  data  unless  sunlight  exposure  is  known.  Actual 
temperatures  of  the  tissues  have  been  taken  by  a  few  observers,  from 

1  See  Freundlich,  H.  Kapillarchemie,  1909,  pp.  504-511,  and  references  given  also  Ostwald, 
W.,  Transl.  by  M.  H.  Fischer.  An  introduction  to  theoretical  and  applied  colloid  chemistry, 
pp.  84-92.  1909. 


Hydration  and  Growth  of  Colloids  and  Cell-masses. 


117 


which  it  is  seen  that  the  cells  may  at  times  have  a  temperature  20°  C.1 
higher  than  the  air.  The  previous  maximum  record  is  that  of  51.5  °C. 
for  Euphorbia  virosa,  obtained  by  Pearson  in  South  Africa  in  1913.2 


40 


30 


20 


10 


\ 


Neon 


12         I         Z3456789 


5a.m.  6        7        6        9        10 


Ss.m.   6 


FIQ.  22. — Temperatures  of  joints  of  Opuntia  in  the  open  at  Desert  Laboratory  on  a  day  in  March 
and  a  day  in  June  1916.  The  solid  line  denotes  air  temperatures,  the  dotted  line  the  temper- 
ature of  a  joint  in  an  east-and-west  position  with  the  faces  north  and  south.  The  broken 
line  gives  the  course  of  the  temperature  of  a  joint  with  its  faces  east  and  west,  and  showing 
a  drop  in  temperature  when  the  margin  is  toward  the  sun  at  midday.  The  upper  part  of  the 
figure  is  compiled  from  the  records  of  March  9,  1916.  The  lower  set  of  lines  is  compiled 
from  the  records  for  June  2,  1916.  Redrawn  after  diagram  furnished  by  Dr.  J.  M.  McGee. 

A  proper  interpretation  of  the  reactions  of  the  plants  used  as  experi- 
mental material  in  the  present  work  might  be  made  only  when  the  ordi- 
nary exposures  were  measured,  and  this  was  undertaken  at  the  request 

1Askenasy,  E.  Ueber  die  Temperature,  welche  Pflanzen  im  Sonnenlicht  annehmen.  Bot. 
Zeitung,  33:441-442.  1875.  See  also  U 'sprung,  A.  Die  physikalischen  Eigenschaften  der 
Laubblatter.  Bibliotheca  Botanica,  12:  (Heft  60),  68.  1903-4. 

2  Pearson,  H.  H.  W.  Observations  on  the  internal  temperatures  of  Euphorbia  virosa  and  Aloe 
dichotoma.  Annals  of  the  Bolus  Herbarium,  vol.  i,  part  2.  Nov.  1914. 


118  Hydration  and  Growth. 

of  the  author  by  Dr.  J.  M.  McGee  at  the  Desert  Laboratory  in  1916. 
Mature  joints  of  Opuntia  were  arranged  vertically  in  two  sets,  one  with 
all  in  a  meridional  position  and  the  other  in  an  east-and-west  position, 
so  that  the  edges  only  were  exposed  to  the  rising  and  setting  sun.  Sets 
of  two  each  were  observed  for  short  periods,  in  which  the  joints  were 
kept  moving  at  a  rate  which  kept  the  edges  or  the  full  surface  exposed 
to  the  direct  rays  of  the  sun  during  the  entire  day.1 

The  course  of  the  temperature  on  two  days  is  shown  in  figure  22. 
The  joints  which  were  in  a  fixed  north-and-south  position  showed  a 
dry  weight  slightly  hi  excels  of  those  which  were  in  an  east-and-west 
position.  The  temperature  of  the  plants  facing  north  and  south  rose 
steadily  until  about  2  p.  m.,  then  began  to  decline,  falling  to  a  point 
below  that  of  the  air  soon  after  sunset.  Joints  facing  east  and  west 
rose  in  temperature  until  about  11  a.  m.,  then  a  slight  drop  took 
place  until  the  sun  shone  on  the  western  face  of  the  joint,  at  which 
time  it  began  to  rise,  reaching  a  maximum  at  4  p.  m.  It  is  evident 
that  the  position  of  these  members  may  through  the  temperature- 
relation  modify  the  rates  of  growth  or  of  hydration.  During  midsum- 
mer the  mature  joints  may  attain  daily  temperatures  of  53°  to  55°  C. 
The  data  obtained  by  the  author  show  that  such  delicate  members  as 
etiolated  shoots,  as  well  as  green  joints,  may  endure  temperature  of 
51.5°  C.  and  to  show  enlargement  a  degree  or  two  below  that  point. 
(See  page  135.) 

The  newly  developed  method  for  determining  temperatures  of  leaves 
by  a  thermo-electrical  method  developed  by  Mrs.  E.  B.  Shreve  at  the 
Desert  Laboratory  promises  to  be  of  great  use  in  the  accurate  deter- 
mination of  this  important  condition  in  plants.2 

A  series  of  measurements  were  made  with  joints  of  Opuntia  grown 
at  the  Coastal  Laboratory,  in  which  the  degree  of  hydration  was  high, 
so  that  the  total  possible  swelling  in  fresh  material  was  small,  although, 
as  may  be  seen  by  reference  to  page  132,  the  increase  of  dried  sections 
was  indicative  of  the  presence  of  a  carbohydrate-protein  complex  of  a 
high  hydration  coefficient. 

Two  recently  matured  joints  grown  in  the  open,  and  subjected  to 
air-temperatures  of  16°  to  22°  C.,  were  taken  for  each  test.  The  sections 
at  the  highest  temperature  had  an  average  thickness  of  9.4  mm.  Those 
used  in  the  tests  at  24°  to  25°  C.  were  about  12  mm.  in  thickness,  and 
the  third  lot  had  an  average  thickness  of  about  the  same. 

Trios  were  placed  hi  glass  dishes  containing  about  25  c.c.  of  liquid, 
the  total  volume  of  the  sections  being  about  3  c.c.  Temperatures  were 
taken  by  mercurial  thermometers,  the  bulbs  of  which  were  thrust  into 
the  dishes  from  time  to  tune.  The  testing-chamber  was  not  illumi- 

1  McGee,  J.  M.  The  effect  of  position  on  the  temperature  and  dry  weight  of  joints  of  Opuntia. 
Rep.  Dept.  Bot.  Research,  Year  Book  No.  15  for  1916,  Carnegie  Inst.  Wash.  1917. 

z  Shreve,  E.  B.  A  thermo-electrical  method  for  the  determination  of  leaf  temperature. 
Plant  World,  22: 100.  1919. 


Hydration  and  Growth  of  Colloids  and  Cell-masses. 


119 


nated,  except  during  the  few  minutes  when  examinations  were  being 
made.    The  swelling  measurements  are  given  in  table  96. 

TABLE  96. 


18  to  20°  C. 

24  to  26°  C. 

44  to  45°  C. 

Distilled  water  

p.  ct. 
11.8 
14 

8.8 
7.3 
12.2 

hours. 
42 
42 

32 
20 

44 

p.  ct. 
13 
14.4 

13.2 
7.8 
13.2 

hours. 
40 
42 

42 
14 
42 

p.  ct. 

7 
8.4 

4.9 
4.9 

5.8 

hours. 
5 
7 

25 
2 
4 

Potassium  nitrate,  0.01  M. 
Potassium   nitrate,   citric 
acid,  0.01  N  

Citric  acid,  0.01  N  

Potassium  hydroxid,  0.01M 

The  data  given  in  table  95  yield  a  number  of  exceedingly  interesting 
suggestions  as  to  the  absorption  and  incorporation  of  water  by  living 
sections  in  the  presence  of  various  substances.  The  time  in  which 
saturation  takes  place  varies  widely  in  the  range  of  temperatures 
which  is  a  normal  one  for  this  plant.  Satisfaction  of  the  water  capacity 
of  the  sections  requires  42  hours  at  the  lowest  temperature  and  but  5 
hours  at  the  highest  in  distilled  water.  The  change  is  from  42  hours 
to  7  in  potassium  nitrate  and  from  32  to  25  in  the  acidified  salt  and  from 
20  to  2  in  the  acid.  The  living  material  already  contains  a  normal 
supply  of  both  substances  and  immersion  in  them  involves  an  increase 
in  their  action.  The  action  of  the  sodium  hydroxid  is,  as  has  been 
found  in  many  instances,  long-continued.  The  maximum  swelling  is 
seen  to  be  accomplished  at  about  25°  C.  The  general  optimum  for  all 
of  the  solutions  is,  however,  probably  above  this  point. 

The  initial  rate  of  increase  at  the  highest  temperature  is  great- 
est, but  it  is  soon  checked.  Etiolated  shoots  of  the  same  species  of 
Opuntia  were  available  and  the  results  of  the  swelling  measurements 
obtained  from  them  afford  some  interesting  comparisons,  although 
it  was  not  possible  to  run  them  as  nearly  parallel  as  might  be  wished. 
These  shoots  were  18  to  25  cm.  long  and  1.5  to  2  cm.  in  width,  some  of 
which  were  hi  a  chamber  at  17°  to  19°  C.  and  others  in  a  chamber  in 
which  their  temperature  ranged  from  30°  to  31°  C.  Swellings  were 
made  at  both  temperatures,  with  the  results  given  in  table  97. 

The  most  striking  feature  of  the  results  is  the  fact  that  the  material 
etiolated  at  the  higher  temperature  shows  a  more  extended  and 
greater  swelling  at  the  lower  temperature.  Unfortunately,  the  fact 
that  the  limiting  effects  of  acidity  on  imbibition  increase  with  rise  hi 
temperature  was  not  known  at  the  time  these  tests  were  carried  out. 

If  the  case  of  the  low-temperature  material  be  taken  up,  it  is  seen 
that  its  increase  is  greatest  at  the  high  temperature  hi  water,  potas- 
sium nitrate,  acidified  potassium  nitrate,  and  acid,  while  the  swelling 
in  alkaline  salt  and  in  hydroxid  is  greatest  at  the  low  temperature. 


120 


Hydration  and  Growth. 


This  fact  suggests  that  the  general  optima  of  the  plants  grown  at  the 
different  temperatures  differ  in  such  manner  that  the  one  grown  at  the 
higher  temperature  has  the  lower  optimum,  and  conversely,  that  the 
one  grown  in  the  cool  chamber  has  its  optimum  at  a  higher  temperature, 
which  is  not  necessarily  at  the  point  at  which  the  tests  were  made. 
The  exceptions  in  the  last  case  in  the  alkaline  solutions  may  afford  a 
clue  to  the  actual  physical  conditions  upon  which  such  differential 

TABLE  97. 


Opuntia  etiolated  at 
17°  to  19°  C. 

Opuntia  etiolated  at 
30°  to  31°  C. 

Swelled  at 
17°  to  16°  C. 

Swelled  at 
30°  to  31°  C. 

Swelled  at 
17°  to  19°  C. 

Swelled  at 
30°  to  31°  C. 

Distilled  water  

p.  ct. 
4.2 
4.8 
4.9 
3.2 

13.7 
10.6 

hra. 
16 
30 
3 
3 

36 
36 

p.  ct. 
7.5 
6.5 
9.5 
4 

9.5 

8 

hrs. 
1.5 
6 
1 
1 

9 
1.3 

p.  ct. 
13.2 
12.7 
7.3 
4.4 

hrs. 
40 
48 
4 
4 

p.  ct. 
3.7 
7.3 
3.7 
3.3 

hra. 
8 
8 
1 
1 

Potassium  nitrate  

Potassium  nitrate,  citric  acid,  0.01  N 
Citric  acid  0.01  N  

Potassium  nitrate,  potassium  hy- 
droxid,  0.01  M  

Potassium  hydroxid,  0.01  N  

action  rests.  According  to  Jenny  Hempel,  the  hydrogen-ion  concen- 
tration of  etiolated  lupine  shoots  was  not  much  different  from  that  of 
normal  green  plants,  although  the  actual  quantity  of  acid  was  much 
greater.  The  amount  of  acid  in  the  plant  grown  at  the  higher  temper- 
ature would,  hi  accordance  with  general  experience,  be  less  than  in 
those  grown  at  the  lower  temperature.1  The  only  suggestions  available 
for  an  explanation  of  the  behavior  in  question  would  rest  upon  the  as- 
sumption that  the  amount  of  acid  was  the  critical  feature,  and  also 
that  the  plants  grown  at  higher  temperature  had  experienced  the  con- 
version of  the  polysaccharids  into  the  pentosans,  which  have  a  rela- 
tively high  coefficient  for  swelling  (see  p.  91). 

It  is  to  be  noted,  in  any  consideration  of  the  action  of  the  colloidal 
systems,  that  fresh  or  living  sections  of  plants  are  already  hi  the  con- 
dition of  the  colloidal  sections  which  have  been  immersed  4  to  8  hours, 
and  that  it  is  the  behavior  of  the  sections  after  they  have  taken  up 
90  per  cent  of  their  total  capacity  for  water  which  comes  into  the 
realm  of  the  living  plant.  Dried  sections  of  plants  come  down  to  a 
water-content  not  much  above  that  of  colloids.  It  also  is  to  be  remem- 
bered that  in  the  advanced  stages  of  the  desiccation  of  cell-masses  the 
protoplasmic  colloids  are  subjected  to  the  action  of  the  dissolved  elec- 
trolytes in  a  concentrated  condition  in  the  final  stages  of  drying.  The 
fixation  of  the  salts  and  acids  which  takes  place  under  these  circum- 


1  Hempel,  J.     Buffer  processes  in  the  metabolism  of  succulent  plants. 
Lab.  d  Carlsberg,  13:  No.  1.     1917. 


Compt.  Rend.  d.  Trav.  d. 


Hydration  and  Growth  of  Colloids  and  Cell-masses.  121 

stances  would  have  such  effect  that  the  hydration  of  the  sections  would 
not  result  in  the  exact  resumption  of  the  former  condition  of  the  cell- 
colloids.  The  field  of  action  of  the  colloid  within  the  limits  mentioned 
offers  a  most  promising  group  of  phenomena,  the  study  of  which  may 
be  expected  to  contribute  in  an  important  manner  to  knowledge  of  the 
mechanics  of  protoplasm. 

Adequate  parallel  measurements  of  the  effects  of  temperature  on 
growth  of  Opuntia  were  not  made,  but  some  records  are  available 
which  show  the  variation  caused  by  the  rise  upon  material  grown  at 
the  lower  temperature  hi  the  above  etiolated  series.  Elongation  by 
growth  of  the  stems  in  question  was  at  the  rate  of  5.2  mm.  daily  at 
16°  to  18°  C.  and  11  to  17  mm.  daily  at  30°  to  32°  C.  The  increase 
amounted  practically  to  a  doubling  for  a  rise  of  10°  C.  The  swelling 
in  transverse  sections  of  similar  material  was  4.9  per  cent  at  17°  to 
19°  C.  and  7.5  per  cent  at  30°  to  31°  C.  in  distilled  water;  and  4.9  per 
cent  at  the  lower  temperature  in  acidified  potassium  nitrate  and  9.5 
per  cent  at  the  higher  temperature.  The  increase  by  swelling  trans- 
versely was  therefore  slightly  less  than  double,  with  a  fair  inference  that 
it  would  have  been  greater  in  the  axis  of  elongation  or  growth.  It  is 
to  be  seen,  therefore,  that  in  the  elongation  of  the  vegetative  axes  of 
plants  the  temperature  effect  is  a  complex  one,  and  that  the  accel- 
erating effect  of  rising  temperature  may  be  primarily  an  increase 
in  absorption  capacity  by  altered  metabolism,  including  lessened 
accumulations  of  acids. 

An  illustration  of  the  failure  of  rising  temperatures  to  increase 
hydration  and  swelling  under  some  conditions  is  furnished  by  the 
behavior  of  sections  of  joints  of  Opuntia  taken  in  a  condition  of  acidosis 
hi  the  morning.  These  increased  no  more  at  27°  C.  than  at  20°  C. 
(see  p.  119).  The  experiences  with  biocolloids  must  be  drawn  upon 
with  care  when  a  parallel  is  sought.  The  sections  of  a  biocolloid 
containing  the  mucilage  from  Opuntia  and  bean  protein  were  seen  to 
swell  2,400  per  cent  at  22°  C.,  while  the  swelling  was  less  than  1,800 
per  cent  at  a  temperature  5  degrees  higher,  but  the  lower  figure  in  this 
case  was  probably  due  to  the  fact  that  the  plates  of  colloidal  material 
began  to  break  up  and  dissolve  out  at  a  lower  stage  of  hydration, 
thus  ending  the  record. 

Various  tests  of  material  were  made  for  the  purpose  of  ascertaining 
the  conditions  prevailing  among  plants  of  different  types.  In  mid- 
April,  sections  were  taken  from  the  terminal  elongation  internodes 
of  Phoradendron  growing  near  the  Desert  Laboratory,  parasitic  on 
Parkinsonia  microphylla.  Such  sections  were  about  3  mm.  in  length 
and  half  that  thickness.  Swelling  was  3  and  5  per  cent  in  distilled 
water  at  20°  and  30°  C.,  respectively,  but  no  appreciable  difference 
could  be  detected  between  the  increases  in  hundredth-no  mal  citric  acid 
at  the  two  temperatures. 


122 


Hydration  and  Growth. 


The  petioles  of  some  young  plants  of  a  Solanum  hybrid  in  the  glass- 
house at  Tucson  were  available  on  April  21,  1918.  Two  series  of  sec- 
tions were  placed  in  distilled  water  and  acid  at  18°  and  38°  C.,  with 
results  shown  in  table  98. 

TABLE  98.  TABLE  99. 


18°  C. 

38°  C. 

p.  ct. 

p.  ct. 

Distilled  water  

4.2 

11.8 

Citric  acid,  0.01  N.  . 

4.2  . 

2.6 

18°  C. 

38°  C. 

a. 

b. 

a. 

6. 

p.  ct. 

p.  ct. 

p.  ct. 

p.  ct. 

Distilled  water  

14 

8 

9.6 

11.7 

Citric  acid,  0.01  N. 

11 

9.7 

6.6 

4.4 

The  swelling  in  distilled  water  was  nearly  three  times  as  great  at  the 
higher  temperature,  while  in  the  acid  solution  a  retardation  took  place 
which  limited  the  total  at  the  higher  temperature  to  something  over 
a  half  that  possible  at  the  lower  point.  The  total  swelling  in  acid  at 
the  lower  temperature  occupied  an  hour  and  at  the  higher  temperature 
it  was  a  matter  of  10  or  15  minutes.  A  similar  speeding-up  of  imbi- 
bition in  water  was  observed.  The  total  capacity  at  the  lower  tem- 
perature was  not  reached  for  8  or  10  hours,  while  at  the  higher  it  was 
something  under  2  hours. 

Plants  of  Phaseolus  which  formed  the  experimental  material  for 
measuring  the  growth  of  pods  and  seeds  bore  some  pods  in  which  the 
beans  were  nearly  mature.  Pods  of  the  same  stage  of  development  as 
one  which  was  under  the  auxograph  for  recording  daily  changes  (see 
p.  156)  were  opened  and  the  unripe  beans  removed.  The  ends  were  cut 
away  and  the  outer  coat  removed.  The  remainder  of  each  cotyledon 
made  one  section,  of  which  three  were  taken  from  separate  pods  for 
swelling.  The  average  thickness  was  3.2  to  3.4  mm.  and  the  swellings 
of  duplicate  series  were  as  given  in  table  99. 

The  higher  temperature  to  which  series  a  was  subjected  appears  to 
be  above  the  point  at  which  maximum  absorption  or  imbibition  takes 
place  in  distilled  water,  as  the  swelling  was  30  per  cent  less  than  at  the 
lower  temperature.  The  retarding  effect  is  much  more  marked  in  the 
acid  solution,  however,  as  the  reduction  of  the  total  capacity  below 
that  shown  at  18°  C.  amounted  to  40  per  cent. 

The  material  in  series  b,  taken  at  a  later  date  and  with  seeds  which 
seemed  to  be  more  nearly  mature,  showed  an  increase  in  swelling  in 
distilled  water  of  about  45  per  cent  over  the  total  at  the  lower  tempera- 
ture, while  the  swelling  in  acid  was  less  than  half  that  at  18°  C.  The 
average  of  the  two  series  is  such  that  the  swelling  in  distilled  water  is 
nearly  the  same  at  both  temperatures,  while  in  acid  the  average  at 
18°  C.  is  10.4  per  cent,  which  is  nearly  double  that  at  38°  C.,  at  which 
point  the  hydration  capacity  seems  to  be  invariably  lower  than  at  the 
lower  temperature.  These  averages  represent  a  total  of  6  cotyledons 
each. 


Hydration  and  Growth  of  Colloids  and  Cell-masses. 


123 


A  final  test  of  variations  in  temperature  upon  material  in  an  acidi- 
fied condition  was  made  with  dried  sections  of  Opuntia.  These  sec- 
tions were  made  by  slicing  away  the  chlorophyllous  layer  from  one 
side  of  the  flat  joint  and  drying  the  remainder  in  the  desiccator  and  in 
sheets  of  blotting-paper  in  such  manner  that  buckling  and  crumpling 
were  prevented.  After  all  of  these  precautions  were  taken,  however, 
the  measurement  of  the  sections  was  subject  to  some  error,  due  to  the 
fact  that  the  fibrovascular  strands  remaining  would  increase  the  thick- 
ness under  the  calipers  without  reacting  in  due  proportion  to  the  action 
of  the  swelling  agent.  A  wide  range  of  figures  was  obtained,  but  it 
was  apparent  that  a  rise  in  temperature  did  not  have  an  effect  on 
material  in  acid  equivalent  to  that  in  distilled  water,  as  will  be  apparent 
from  the  measurements  obtained  from  sections  which  were  0.43  to 
0.46  mm.  in  thickness  (table  100). 

TABLE  100. 


Swelling  at 
18°  C. 

Aver- 
ages. 

Swelling  at 
28°  C. 

Swelling  at 
38°  C. 

Aver- 
ages. 

Distilled  water  

p.  ct.      p.  ct.      p.  ct. 
315        385        486 

p.  ct. 
395 

p.  ct. 
453 

p.  ct.      p.  ct. 
500        413 

p.  ct. 

457 

Citric  acid,  0.01  N  

360        430        460 

417 

460 

477        400 

439 

The  increase  in  swelling  in  distilled  water  is  seen  to  be  about  twice 
that  in  the  acid  in  the  rise  from  18°  C.  to  38°  C.  The  influence  which 
the  condition  in  question  may  exert  on  the  rate  of  growth  is  obvious. 
Thus  the  course  of  enlargement  of  an  organ  or  of  a  cell-mass,  in  so 
far  as  this  consists  in  hydration,  may  vary  widely  in  the  first  instance, 
because  of  the  residual  acids  in  the  colloids,  and  the  balance  or  accumu- 
lation of  this  will  in  turn  depend  upon  the  effect  of  the  enzymic  or 
respiratory  processes  in  metabolism.  It  is  obvious  that  a  rise  of  10 
degrees  from  the  customary  morning  temperature  of  15°  C.,  which  has 
accompanied  so  many  of  these  experiments,  might  result  in  an  ac- 
celeration of  growth  determined  by  the  reduced  acidosis  of  the  plant. 
A  rise  from  the  same  temperature  later  in  the  day  or  under  other  con- 
ditions of  illumination  would  necessarily  have  a  different  result.  An 
extension  of  the  attempts  to  bring  rates  of  growth  into  a  figure  or 
formula,  therefore,  would  be  a  forced  application  of  knowledge  of  one 
process  to  a  complicated  procedure  which  results  in  no  positive  advance. 
Variation  of  temperature  results  in  modification  of  the  rate  of  enzymic 
processes  and  of  the  forms  of  metabolism  included  under  and  associated 
with  respiration,  in  modification  of  the  rate  of  absorption  of  water  by  the 
organism  from  its  medium  or  substratum,  and  modification  of  the  water- 
holding  capacity  of  the  cell-colloids  after  a  mode  determined  by  their 
carbohydrate-protein  ratio  and  by  their  state  of  acidosis.  The  actual 
increase  in  volume,  will  also  be  influenced  to  some  extent  by  the  continual 
water-loss  from  the  surface. 


Hydration  and  Growth. 

The  conception  of  a  temperature  coefficient  of  growth  must  be  taken 
as  an  integration  of  the  action  of  a  constellation  of  forces  acting  upon 
colloidal  material  of  varying  constitution.1  The  agencies  in  question 
do  not  run  parallel  in  their  effects  and  interlock  hi  the  most  intricate 
manner.2  It  is  not  surprising,  therefore,  to  find  that  the  coefficient  of 
temperature  as  applied  to  growth,  which  is  usually  calculated  in  terms 
of  relative  effect  for  each  variation  of  10°  C.,  has  but  little  usefulness, 
except  between  10°  and  30°  C.2  The  value  of  Q10  as  it  is  usually  written 
varies  between  1.12  and  5  or  6,  and  the  variation  in  any  given  organism 
is  usually  very  great  above  30°  or  35°  C.  Thus,  in  my  own  experi- 
ments with  Opuntia,  the  actual  range  of  growth  was  found  to  extend 
from  as  low  as  7°  C.  under  some  circumstances  to  51.5°  C.  under  others, 
but  no  single  individual  was  seen  to  grow  throughout  this  range.  The 
combination  of  conditions  which  would  enable  it  to  do  so  are  not  likely 
to  occur  in  a  state  of  nature  and  would  be  difficult  to  bring  about 
experimentally. 

In  addition  to  the  complexities  of  interplay  of  molecular  forces  to  be 
reckoned  with — and  they  appear  most  formidable  to  the  chemist 
familiar  with  their  nature — the  application  of  ratios  or  formulae  to 
variations  in  growth  produced  by  temperature  in  the  higher  plants 
encounters  still  other  difficulties.  Chief  among  these  is  the  fact  that 
the  growing  region  of  a  plant  may  vary  in  actual  and  relative  amounts 
of  embryonic  cell-masses  and  of  fixed  non-expanding  tissues.  External 
measurements  of  elongation,  even  when  applied  to  root-tips,  may  have, 
in  consequence,  but  doubtful  value. 

A  brief  description  has  been  given  on  page  96  of  the  unsatisfied 
water  capacity  of  the  conns  of  Brodicea,  from  the  apices  of  which  two 
or  three  leaves  20  to  30  cm.  long  arise  and  elongate  by  the  action  of  a 
mass  of  embryonic  cells,  which  maintain  a  basal  position  during  the 
entire  development  of  the  leaf.  The  corms  habitually  lie  5  to  10  cm. 
below  the  surface  of  the  soil  and  the  growing  region  operates  under  the 
influence  of  the  soil,  not  the  air,  temperature.  It  was  therefore  ar- 
ranged to  grow  some  of  these  plants  hi  pots,  taking  the  temperatures 
by  thermometers  thrust  into  the  soil,  hi  the  vicinity  of  the  bulbs.  Such 
cultures  in  small  chambers  with  thermostatic  control  yielded  some 
data  of  interest.  An  attempt  was  made  to  ascertain  the  relative  rates 
of  increase  or  amount  of  water  which  might  be  absorbed  by  the  corms 
and  by  the  growing  regions  of  the  leaves  at  different  temperatures. 
A  trio  of  corms,  each  of  which  consisted  of  the  older  basal  corm  and 
the  recently  formed  younger  one,  having  an  average  height  of  12  mm., 
were  at  first  swelled  to  saturation  at  19°  C.  and  after  two  days,  when 
quiescence  had  been  reached,  the  preparation  was  placed  in  a  warmer 

1  Osterhout,  W.  J.  V.     Some  aspects  of  the  temperature  coefficients  of  life-processes.     Jour. 
Biol.  Chem.,  32:  No.  1,  p.  23.     1917. 

2  See  Barry,  F.     The  influence  of  temperature  upon  chemical  reactions  in  general.     Amer. 
Jour.  Bot.,  1:  203-225.     1914. 


Hydration  and  Growth  of  Colloids  and  Cell-masses. 


125 


chamber,  where  the  water  in  which  they  were  immersed  was  raised 
to  28°  C.  and  kept  at  that  point  for  40  hours.  Increased  imbibition 
ensued,  which  resulted  in  an  elongation  of  1  mm.  or  8  per  cent  of  their 
total  height.  Measurements  of  the  growth  of  leaves  arising  from  the 
apices  of  such  corms  showed  that  they  maintained  a  rate  of  about  0.25 
mm.  per  hour  at  air-temperatures  of  19°  C.,  which  was  increased  to  0.49 
mm.  per  hour  at  27°  to  28°  C.;  the  temperature  coefficient  of  such 
elongation  would  thus  be  nearly  2.5. 

A  second  pair  of  preparations  with  young  leaves  about  10  to  12  cm. 
long  was  placed  in  a  small,  well-lighted  thermostat,  and  the  apices  of 
the  second  or  younger  leaf  were  attached  to  the  auxographs.  The 
action  of  these  plants  was  followed  for  a  period  of  about  20  days, 
during  which  time  the  leaves  reached  a  length  of  25  to  30  cm.  The 
growing  region  remains  basal  to  the  leaf  and  the  rate  of  growth  during 
the  course  of  development  is  much  flatter  than  in  stems,  presenting 
some  of  the  features  of  root-tips. 

Three  objects  were  in  view  in  the  measurements  of  the  rates  of 
elongation:  (1)  estimation  of  the  rate  in  alterations  from  a  low  to  a 
high  temperature  and  vice  versa;  (2)  accelerations  due  to  changes  from 
one  temperature  to  another;  (3)  the  effects  of  small  and  of  wide  vari- 
ations in  temperature. 

The  principal  data  concerning  the  experiment  are  given  in  table  101. 

TABLE  101. 


No.  1 

No.  2 

Rates 
per  hour 
for— 

At  tempera- 
tures of  — 

Rates 
per  hour 
for— 

At  temera- 
tures  of  — 

mm. 

hrs. 

°C. 

mm. 

hrs. 

•C. 

0.24 

24 

31-33 

1.7 

12 

25-27 

0.1 

24 

9.5-11 

0.11 

24 

9.5-11 

1.1 

18 

20.5-21.5 

0.8 

15 

19.5-21 

1.4 

7 

20-21 

1.1 

7 

19-20 

0.3 

11 

9-10 

0.4 

11 

9-10 

1.2 

8 

19.5-20 

0.9 

8 

19-20 

0.6 

12 

8-9 

0.27 

12 

8-9 

1.4 

4.5 

27-28.5 

1.22 

4.5 

26-27.5 

0.3 

10 

7 

0.3 

10 

7 

1.25 

6 

20-21 

0.8 

6 

19-20 

2.25 

8 

28 

0.9 

5 

30-31 

0.7 

5 

33 

0.7 

9 

20-21 

0.7 

9 

23 

0.3 

5 

17 

0.15 

5 

17 

0.8 

6 

27-28 

0.27 

15 

30-31 

0.2 

15 

17 

0.2 

15 

17 

0.5 

28 

17 

0.5 

28 

17 

1.1 

15 

26-28 

1.7 

7 

26-28 

0.7 

12 

17-18 

0.6 

16 

16-17 

The  maximum  rate  displayed  was  at  a  temperature  slightly  under 
30°  C.,  varying,  of  course,  with  the  preceding  experience.  Rising 
temperatures  are  seen  to  accelerate  growth  with  a  coefficient  slightly 


126  Hydration  and  Growth. 

above  2,  except  in  cases  in  which  the  rise  carried  the  temperature  to 
30°  C.  or  above  and  also  when  the  change  was  as  much  as  20°  C.  In 
the  earlier  stage  of  development  the  rate  rose  from  0.1  mm.  per  hour  at 
11°  C.  to  0.8  mm.  per  hour  at  21°  C.,  and  in  another  leaf  from  0.1  mm. 
to  1.1  mm.  per  hour  in  passing  from  9°  to  20°  C.  A  change  from  9° 
to  27°  C.  resulted  in  a  decreased  rate  in  one  case,  while  in  others  rais- 
ing the  temperature  from  27°  or  28°  to  33°  C.  had  no  effect. 

Falling  temperatures  were  accompanied  by  reductions,  with  a  usually 
lower  coefficient,  it  being  noticeable  that,  as  exceptions,  the  rate  in 
one  case  decreased  from  0.7  mm.  per  hour  at  23°  C.  to  0.15  mm.  per 
hour  at  17°  C.,  while  in  another  case  the  rate  decreased  from  0.8  mm. 
per  hour  at  27°  C.  to  0.2  mm.  at  17°  C.  This  unusual  reduction  is 
ascribed  to  the  fact  that  the  door  of  the  thermostat  was  opened  to 
facilitate  the  cooling,  which  resulted  in  the  exposure  of  the  leaves  to 
low  relative  humidity  during  the  greater  part  of  this  period. 

Closing  the  chamber  and  continuing  the  temperature  of  16°  to  17°  C. 
for  28  hours  longer  gave  a  record  in  which  the  rate  gradually  rose  from 
the  low  figure  mentioned  to  0.5  and  0.6  mm.  per  hour,  which  was  nearest 
the  expectancy  in  comparison  with  the  rate  of  0.7mm.  per  hour  at 
23°  C.  which  had  been  displayed  in  the  previous  period. 

In  the  following  period  a  rise  of  temperature  from  17°  to  26°  or 
28°  C.  was  accompanied  by  accelerations  of  0.5  to  1.7mm.  and  from 
0.5  to  1.1  mm.  per  hour.  The  reversal  of  the  temperatures  brought  the 
rates  down. to  0.6  mm.  from  1.7  mm.  in  a  change  from  28°  to  16°  or  17° 
C.  and  from  1.1  mm.  to  0.7  mm.  in  a  change  from  26°-28°  to  17°-18° 
C.  No  other  variations  were  observed  in  the  three  weeks  in  which 
these  organs  were  under  observation  which  could  be  ascribed  simply 
to  the  change  of  temperature.  Usually  the  change  in  the  thermostatic 
temperature  would  be  followed  within  an  hour  or  two  by  that  of  the 
soil  around  the  bulbs  and  the  result  would  be  a  gradual  adjustment  of 
the  rate,  increasing  or  decreasing,  with  no  breaks  or  erratic  features, 
in  the  tracings  which  gave  a  continuous  records  of  the  lengths. 

In  conclusion,  it  is  to  be  pointed  out  that  the  foregoing  observations 
show  that  the  hydration  capacity  of  cell-masses  may  bear  some  re- 
lation to  the  temperature  at  which  they  are  formed  and  under  which 
they  have  functioned  for  some  time,  and  also  that  the  unsatisfied  water 
capacity  of  a  tissue  will  be  affected  by  the  relation  between  absorption 
and  water-loss  by  transpiration.  Some  gross  measurements,  which 
have  been  made  with  an  accuracy  quite  adequate  for  the  determination 
of  the  point  in  question,  show  that  plants  in  the  tropics  show  a  rate  of 
growth  which  may  be  directly  correlated  with  relative  humidity  and 
the  transpiration  to  be  inferred. 

Retardation  of  growth  of  an  organ  of  a  higher  plant  may  be  the 
result  of  such  direct  water-loss,  or,  on  the  other  hand,  it  may  be  directly 
connected  with  the  lessening  of  the  water  capacity  of  colloidal  masses 


Hydration  and  Growth  of  Colloids  and  Cell-masses.  127 

under  acid  conditions,  as  has  been  demonstrated  both  in  sections  of 
pentosan-protein  biocolloids  and  in  living  and  dried  sections  of  plants. 
The  application  of  a  temperature  coefficient  derived  from  a  simple 
equation  to  the  rates  of  growth  6f  organs  of  green  plants  has  a  certain 
practical  value  when  temperatures  between  the  minimum  and  the 
maximum  rate  are  under  consideration.  As  changes  in  temperature 
affect  a  number  of  constituent  processes  in  growth,  including  absorp- 
tion and  diffusion,  transpiration,  adsorption,  action  of  acidity  or 
hydrogen-ion  concentration,  formation  of  the  amino-acids,  enzymic 
action  and  oxidations,  and  all  transformations  of  the  carbohydrates, 
the  empirical  character  of  such  indices  must  be  kept  well  in  mind. 
The  character  of  temperature  coefficients  is  sufficiently  indicated  by 
the  fact  that  they  are  not  found  to  apply  through  the  range  of  practi- 
cable or  habitual  conditions  of  the  organism  and  that  the  values  change 
within  the  range  of  8°  to  30°  C.  when  falling  and  rising  temperatures 
are  contrasted. 


X.  IMBIBITION  AND  GROWTH  OF  OPUNTIA. 

Living  cells  and  colloidal  masses  are  never  in  a  state  of  perfect 
equilibrium  with  the  environment,  and  the  possible  adjustments  in 
growing  cells  in  which  the  colloidal  substances  are  increasing,  changing 
in  constitution,  and  varying  in  condition,  may  be  great  both  as  to 
velocity  and  amplitude.  Artificially  compounded  colloids,  such  as 
the  agar-protein  mixtures  which  have  been  so  freely  used  in  this  work, 
show  a  similar  delicate  series  of  adjustment  and  correlations  as  they 
pass  from  the  dry  state  to  one  of  almost  completely  satisfied  hydration; 
but  when  they  reach  this  stage  they  are  in  the  general  condition  of 
mature  cells  or  tissues  and  then  show  only  the  minute  adjustments 
which  follow  the  modifications  of  the  environment  very  closely. 
Living  cell-masses  are  to  be  considered  as  masses  of  colloid  the  inti- 
mate substance  of  which  is  being  constantly  altered  by  metabolism 
and  by  the  incorporation  of  new  material.  The  actual  capacity  of  a 
gel  for  water  and  its  consequent  state  of  swelling  may  be  practically 
satisfied  at  any  stationary  temperature.  Such  equable  temperatures 
do  not  occur  in  natural  environments  and  may  be  observed  only  in 
control  chambers.  The  pen  of  the  instrument  employed  in  methods 
of  accurate  measurement  of  a  hydrated  mass  of  colloid  indicates  con- 
stant variations  in  volume,  due  to  the  solvation  or  dispersion  of  some 
of  the  mass  in  the  water  in  which  it  may  be  immersed. 

The  swelling  of  a  cell-mass  is  to  be  considered  as  determined  by  the 
hydration  capacity  of  its  colloids  at  the  beginning  of  immersion  plus 
whatever  additional  capacity  or  variation  may  be  developed  by  the 
rearrangement  of  its  material,  reformation  of  its  compounds,  and 
migrations  of  its  molecular  aggregates.  Of  the  increased  volume  char- 
acterized as  growth,  98  per  cent  results  from  hydration.  The  grow- 
ing cell-mass,  however,  in  addition  to  the  initial  hydration  capacity 
of  its  mass,  is  continually  adding  material  which  has  hydration  ca- 
pacity, and  metabolic  activities  result  in  the  accumulation  of  acids 
and  other  substances  which  affect  the  coefficient  of  swelling.  The 
major  procedure  in  growth  would  theoretically  be  imitated  if  minute 
particles  of  powdered  colloid  could  be  continuously  introduced  into  a 
swelling  mass. 

Measurements  of  hydration  in  plants  were  made  with  disks  about 
12  mm.  across,  cut  from  the  flattened  joints  of  an  opuntia,  which 
ranged  from  5  to  20  mm.  in  thickness  (fig.  23).  Such  sections 
consisted  of  the  indurated  epidermal  layers,  between  which  was  a 
cylindrical  mass  of  parenchymatous  cells,  the  outer  ones  being  chlo- 
rophyllose.  An  anastomosed  network  of  thin,  fibrovascular  strands 
was  included  in  the  parenchymatous  mass,  and  this  mechanical  tis- 
sue checked  expansion,  so  that  care  was  necessary  not  to  include  the 
larger,  firmer  strands  in  the  section.  Three  of  such  disks  about  12  mm. 

128 


Imbibition  and  Growth  of  Opuntia. 


129 


across  the  epidermal  surface  and  from  6  to  11  mm.  in  thickness  were 
arranged  in  a  triangle  in  the  bottom  of  a  Stender  dish  and  a  triangle  of 
thin  sheet-glass  arranged  to  rest  its  apices  on  the  three  disks  (see  fig.  1) 
The  vertical  swinging  arm  of  an  auxograph  was  now  adjusted  to  a 
shallow  socket  in  the  center  of  the  glass-  triangle,  while  the  pen  was  set 
at  zero  on  the  recording  sheet.  Water  or  a  solution  being  poured  into 
the  dish,  the  bulb  of  a  thermometer  was  adjusted  in  it  and  the  course 
of  the  swelling  was  traced,  the  record  showing  the  average  result  of 
the  action  of  the  trio  of  specimens.1 


FIG.  23. — Calipers  used  in  obtaining  thickness  of  trio  of  sections. 
Swelling  is  calculated  on  average. 

Extensive  records  of  the  growth  of  the  flattened  joints  of  certain 
platyopuntias  having  been  made  at  the  Desert  Laboratory,  and  as  a 
series  of  analyses  of  the  carbohydrate  constituents  of  these  plants  was 
being  carried  out,  parallel  measurements  were  planned  which  might 
show  possible  connection  between  the  composition  of  these  members 
and  their  imbibition  or  swelling  reactions.  The  first  series  began  with 
the  young  joints  which  are  formed  in  April  and  May,  reaching  maturity 
in  about  40  or  50  days  and  extending  through  the  seasons,  including 
the  dry  foresummer,  the  summer  rainy  season,  then  the  dry  after- 
summer,  merging  into  the  winter  with  its  final  rainy  season.  The  im- 
bibition capacity  of  sections  taken  from  the  joints  ran  a  course  shown 
in  table  102. 

1  MacDougal,  D.  T.  Imbibitional  swelling  of  plants  and  colloidal  mixtures.  Science,  44:  502- 
505.  1916.  Also  MacDougal.  Mechanism  and  conditions  of  growth.  Mem.  N.  Y.  Bot.  Gar- 
den, 6: 5-26.  1916. 


130 


Bydration  and  Growth. 
TABLE  102. — Swelling  of  joints  formed  in  1916. 


Water. 

HC1,  0.01M. 

NaOH, 
0.01M. 

May  18,  1916  

p.  ct. 
24.3 

p.  ct. 
30  0 

p.  ct. 
40  0 

June  2,  1916  

23.6 

16  4 

22  9 

Nov.  2,  1916  

20.5 

21  0 

22  2 

Nov.  23,  1916  

48  0 

45  4 

35  3 

Jan.  24-25,  1917  (12  sections).  .  . 
Feb.  20-21,  1917  (6  sections)  
Mar.  23-24,  1917  (6  sections)  .  .  . 
Apr.  24,  1917  

25.7 
10.7 
9.4 
21.8 

27.9 
11.7 
12.0 
20.4 

25.0 
10.8 
10.9 
13  9 

No  record  was  made  of  the  temperatures  of  the  swelling  sections, 
but  these  in  general  were  determined  by  the  seasonal  conditions. 
Thus,  sections  were  swelled  in  November  at  16°  to  18°  C.  and  at  25° 
to  28°  C.  in  April  and  May. 

The  measurements  given  in  table  102  were  made  primarily  for  the 
purpose  of  following  the  changes  in  the  flattened  stems  of  Opuntia  as 
they  go  toward  maturity.  The  joints,  having  reached  the  mature  con- 
dition in  November,  the  changes  for  the  next  4  or  5  months  are  not 
directly  connected  with  growth,  although  the  action  of  living  cells  is 
concerned.  The  period  of  late  summer  is  one  of  progressive  desic- 
cation, in  which  the  water-loss  is  much  greater  than  the  supply 
obtained  from  the  soil,  and  this  drying  out  continues  until  some  time 
in  January  and  February,  when  the  winter  rains  saturate  the  soil. 
It  would  be  expected  that  if  sections  of  the  partially  desiccated 
plants  were  taken  during  the  dry  period,  the  swellings  which  they 
would  show  when  placed  in  solutions  would  be  a  direct  function  of 
the  net  water-loss  which  they  had  sustained,  rather  than  of  changes 
in  composition.  It  was  therefore  necessary  to  devise  a  method  by 
which  the  material  could  be  reduced  to  a  standard  in  which  the 
seasonal  water-condition  would  be  eliminated.  This  was  accom- 
plished by  the  use  of  dried  slices  from  the  median  portion  of  the 
joints  in  such  manner  as  to  exclude  nodes  and  spines. 

The  joints  were  laid  flat  on  a  table  and  against  a  strip  of  wood  5 
or  6  mm.  in  thickness,  which  served  as  a  guide  for  sliding  a  knife  to 
cut  away  one  epidermal  region.  This  being  done,  the  piece  was  turned 
over  and  a  horizontal  movement  of  the  knife  along  the  lowered  guide 
would  cut  away  the  other  epidermal  region,  leaving  a  slice  3  to  5  mm. 
in  thickness,  which  was  dried  in  the  air  at  temperatures  of  15°  to  18°  C., 
using  filter-paper  or  blotting-paper  to  keep  the  sections  plane  during  a 
part  of  the  process.  Sections  cut  from  the  dried  pieces  were  calibrated 
by  scales  of  the  type  used  commercially  for  measuring  the  thickness 
of  paper  (fig.  8),  and  then  swelled  under  the  auxograph  in  the  usual 
manner.  The  calculation  of  the  percentage  of  swelling  on  the  final 
thickness  of  the  dried  material  eliminated  the  factors  attendant  upon 


Imbibition  and  Growth  of  Opuntia. 


131 


the  condition  of  the  fresh  material.     It  is  important  that  the  thick- 
ness of  the  slices  before  drying  be  approximately  equivalent. 

The  measurements  obtained  from  living  sections  inclusive  of  the 
entire  thickness  of  joints  and  from  dried  slices,  given  in  table  103, 
illustrate  the  seasonal  variations  in  mature  stems. 

TABLE  103. — Swelling  of  mature  joints  at  different  seasons. 


Date. 

Solutions. 

Water. 

Citric 
acid, 
0.01  N. 

Sodium 
hydroxid, 
0.01  M. 

Potassium 
chloride, 
hydro- 
chloric 
acid, 
0.01  M. 

Oct.  1917  (living  sections)  (18-25°  C.)  

p.  ct. 
98 
277 
214 
194 
55 
143 
20 
238 
4.8 
550 
37 
304 
54 
195 

p.  ct. 
116 
300 
238 
317 
80 
143 
14 
210 
5.5 
463 
40 
322 
51.6 
166 

p.  ct. 
100 
177 

p.  ct. 

Dec.  1917  (dried  slices)  (16-18°  C.)  

Jan.  3,  1918  (living  sections)  (16-18°  C.)  

(Dried  slices)  (12-14°  C.)  

282 
85 
143 
14 
210 
5.7 
600 
36.5 
310 
50.3 
208 

Jan.  24,  1918  (living  sections)  

(Dried  slices)  (16-18°  C.)  

March  4,  1918  (living  sections)  

14.3 
210 
3.5 
388 
37 
274 
42.7 
230 

(Dried  slices)  (18-20°  C.)  

March  30,  1918  (living  sections)  

(Dried  slices)  (20-22°  C.)  

Apr.  25,  1918  (living  sections)  

(Dried  slices)  (23°  C.)  

May  28,  1918  (living  sections)  

(Dried  slices)  (20-25°  C.)  

The  continued  dry  condition  is  seen  to  result  in  desiccation,  which, 
of  course,  is  followed  by  increased  hydration  when  such  living  sections 
are  swelled,  the  maximum  effect  being  reached  in  January,  before  the 
beginning  of  the  winter  rains. 

The  tests  of  the  dried  sections  which  show  the  swelling  after  the 
colloids  have  been  subjected  to  the  action  of  concentrating  salts  have 
a  value  derived  from  the  fact  that  all  are  reduced  to  an  equivalent 
water-content,  accompanied  by  irreversible  coagulations  of  the  more 
complex  proteins.  Although  we  here  introduce  a  new  set  of  conditions, 
the  effects  of  which  are  not  easily  to  be  evaluated,  yet  it  is  none  the 
less  significant  that  the  entire  series  of  preparations  reach  their 
maximum  water  capacity  at  the  end  of  March.  Since  some  changes 
may  have  been  brought  about  in  the  proteins,  it  is  important  to 
follow  the  course  of  the  carbohydrates. 

The  variation  in  the  sugar-content  is  best  illustrated  by  the  data 
given  in  table  104,  as  determined  by  Dr.  Spoehr.1 

It  is  clear  that  the  colloidal  material  of  the  cell-mass  of  this  plant 
does  not  come  to  a  condition  of  highest  imbibition  capacity  at  the  end 
of  the  period  of  desiccation  in  the  season  of  low  temperature,  but  the 
maximum  is  found  after  the  beginning  of  a  season  of  rising  temperatures 
and  of  accumulating  sugars,  coupled  with  an  inadequate  water-supply. 

Spoehr,  H.  A.    The  pentoae  sugars  in  plant  metabolism.    The  Plant  World,  121 :  365-379.    1917. 


132 


Hydration  and  Growth. 


The  extended  measurements  of  the  swelling  of  biocolloids  containing 
sugars,  dextrose  and  sucrose,  and  of  such  mixtures  in  solutions  of 
sugars,  show  that  these  have  but  little  influence  upon  imbibition  capac- 
ity. These  results  suggest  that  it  is  to  the  variations  in  the  pentoses 
as  represented  by  the  mucilages  of  the  opuntias  that  we  must  look  for 
a  part  of  the  varying  imbibition  capacity  of  these  and  perhaps  other 
plants  as  well. 

TABLE  104. — Seasonal  variations  in  sugar-content  of  Opuntia  sp. 


Date. 

July 
5. 

July 
31. 

Sept. 
20. 

Oct. 
27. 

Nov. 
15. 

Dec. 
20. 

Jan. 
11. 

Feb. 
16. 

Mar. 
17. 

April 
25. 

May 
22. 

Dry  weight  

36.38 

16.45 

19.66 

20.30 

23.05 

30.10 

22.20 

22.33 

19.50 

24.30 

25  25 

Total  sugars,  p.  ct  

20.03 

13.24 

18.44 

20.90 

18.75 

28.95 

19.10 

21.32 

28.05 

32.40 

30  15 

Total  hexose  sugars,  p.  ct.  .  . 
Total  pentose  sugars,  p.  ct  .  . 
Pentosans,  p.  ct  

10.45 
9.26 
9.04 

8.60 
4.39 

8.83 
9.08 
8.86 

9.32 
10.95 
10.47 

5.50 
12.50 
11.35 

7.90 
10.45 
10.10 

14.95 
4.73 
4.40 

14.90 
6.07 
5.51 

22.16 
5.55 
4.75 

22.70 
9.15 

8  68 

17.08 
12.34 
12  17 

Pentoses,  p.  ct  

0.20 

0.24 

0.48 

0.82 

0.35 

0.43 

0.65 

0.82 

0  48 

0  16 

Ratio  of  total  pentose  sug- 
ars to  total  sugars  

0.462 

0.332 

0.492 

0.624 

0.667 

0.551 

0.248 

0.283 

0.198 

0  283 

0  409 

Ratio  of  total  hexose  sug- 
ars to  total  sugars  

0.522 

0.650 

0.479 

0.446 

0.293 

0.417 

0.783 

0.698 

0  791 

0  702 

0  567 

The  vegetative  conditions  at  the  Coastal  Laboratory  are  widely 
different  from  those  at  the  Desert  Laboratory,  at  which  the  above 
results  were  obtained.  Sections  of  fresh  material  at  the  end  of  August 
at  the  first  place  showed  swellings  of  7  to  10  per  cent  at  tempera- 
tures of  15°  to  16°  C.  This  was  characteristic  of  the  end  of  the  sum- 
mer cool  and  foggy  season.  Higher  temperatures  and  greater  average 
daily  illumination  resulted  in  increased  desiccation  during  August  and 
September,  with  the  result  that  living  sections  showed  an  unsatisfied 
imbibition  capacity  of  18  per  cent  at  15°  to  16°  C.  The  method  of 
preparing  dried  median  slices  was  developed  at  this  time,  and  these 
showed  swellings  of  500  per  cent  at  15°  C.  and  570  per  cent  at  20°  C. 
The  total  proportion  of  pentose  sugars  at  this  time  was  19.10  per  cent 
as  compared  with  4.39  per  cent  at  Tucson,  calculated  on  dry  weight. 
Dried  sections  at  the  Coastal  Laboratory  taken  at  the  end  of  Janu- 
ary, after  2  months  of  the  cool  season  but  with  a  fair  supply  of 
moisture,  were  found  to  swell  300  per  cent  at  14°  to  16°  C.  A  test 
was  also  made  of  sections  comprising  only  the  epidermal  and  chloro- 
phyllose  layers  of  the  same  material,  and  these  were  found  to  show 
an  increase  of  262  per  cent  at  the  same  temperature. 

Decrease  in  swelling  capacity  is  seen  to  occur  in  the  cool  season  at 
Tucson  and  at  Carmel.  The  composition  of  the  joints  in  September, 
representing  approximately  the  condition  of  the  material  at  both 
places  in  October,  as  determined  by  Dr.  H.  A.  Spoehr,  is  shown  by 
table  105.1 

1  MacDougal  and  Spoehr.    Growth  and  imbibition.    Proo.  Am.  Phil.  So*.,  66: 289.  1917. 


Imbibition  and  Growth  of  Opuntia. 


133 


TABLE  105. 


Cannel. 

Tucson. 

Water  

p.  ct. 
91.15 

p.  ct. 
80.34 

Total  sugars  

2.61 

4  30 

Total  polysaccharides  
Hexose  polysaccharides.  .  . 
Disaccharides  

1.94 
.09 
.07 

3.50 
1.65 
04 

Hexoses  

.52 

06 

Pentoses  

.14 

05 

Pentosans  

1  70 

1  74 

The  material  to  which  the  high  hydration  capacity  of  Opuntia  is  due 
is  the  mucilage  which  exudes  from  the  damaged  cells  of  cut  surfaces 
of  the  parenchymatous  tissues.  This  is  included  among  the  pentosans 
together  with  agar  and  various  gums,  including  tragacanth,  acacia, 
cherry  gum,  and  prosopis  gum,  many  of  which  have  a  high  hydra- 
tion capacity  and  infinite  dispersion  in  water. 

The  manner  of  the  formation 
of  this  material  is  a  matter  of 
no  little  importance  in  connec- 
tion with  varying  growth  and 
hydration  capacity.  Briefly 
stated,  the  depletion  of  the 
water-content  of  a  cell  accel- 
erates the  conversion  of  poly- 
saccharids  with  low  hydration 
capacity  into  pentosans  (muci- 
lages and  gums)  which  have 
an  extremely  high  water  capac- 
ity, and  it  is  this  change  which  is  followed  by  the  large  swelling  of 
tissues  in  certain  stages  or  in  dry  seasons.  The  transformation  in 
question  is  not  reversible.  While  some  decrease  in  these  pentosans 
may  occur,  it  is  not  known  by  what  steps  it  takes  place.1 

The  composition  of  etiolated  plants  presents  proportions  of  the 
principal  constituents  different  from  those  of  the  green  plant,  and  if 
one  of  the  main  conclusions  of  the  present  work  is  valid,  they  might 
be  expected  to  exhibit  some  water-relations  different  from  those  of 
green  plants. 

Some  shoots  of  Opuntia  which  had  developed  in  a  dark  room  at 
Carmel  in  equable  temperatures  below  20°  C.  were  available  in  July 

1917,  and  these  displayed  some  singular  temperature  effects.    The 
stems  were  from  20  to  30  cm.  long  and  compressed  ovate  in  cross- 
section.     Short  sections  were  cut  out  and  immersed  in  dishes  in  the 
usual  manner.     Most  of  the  development  had  been  at  16°  to  18°  C., 
and  some  of  the  shoots  were  placed  in  a  second  chamber  at  30°  to 
31.1°  C.  for  2  days  before  being  swelled.    The   illumination  of  a 
small  incandescent  bulb  used  in  obtaining  these  measurements  had 
given  the  stems  a  slight  greenish  tinge,  but  the  energy  derived  from 
this  source  was  not  sufficient  to  have  caused  any  serious  change  in 
the  composition  of  the  shoot.     Sections  were  about  5.5  mm.  in  dia- 
meter, and  sets  of  three  were  selected  for  testing  under  the  auxo- 
graph,  so  that  the  older  basal  parts  of  the  stem  and  the  newest 
apical  portions  were  represented  in  the  separate  measurements.    One 

1  See  MacDougal  and  Spoehr.     The  origination  of  xerophytism.     The  Plant  World,  21 :  245. 

1918.  Also  MacDougal,  Richards,  and  Spoehr.     The  basis  of  succulence  in  plants.     Bot.  Gaz., 
67: 405.     1919. 


134  Hydration  and  Growth. 

series  was  swelled  at  the  temperature  of  30°  to  31°  C.,  at  which  the 
shoot  had  stood  for  several  days.  The  second  series  was  swelled  at 
17°  to  19°  C.,  in  which  equivalent  shoots  stood,  which  were  to  be 
used  as  a  comparison.1 

The  most  remarkable  feature  of  these  measurements  is  that  in 
which  the  material  grown  at  30°  to  31°  C.  for  two  days  showed  a  greater 
increase  when  swelled  at  a  lower  temperature  (see  p.  119).  The  entire 
set  of  reactions  is  indicative  of  a  colloidal  complex  of  different  con- 
stitution from  that  of  green  plants. 

Analyses  necessary  to  establish  the  nature  of  such  divergent  con- 
stitution of  etiolated  opuntias  could  not  be  made,  but  data  obtained 
from  other  species  grown  in  darkness  are  available.  The  results  of 
Palladin  show  that  stems  of  such  plants  have  less  nitrogen  than  nor- 
mal green  plants,  and  all  workers  who  have  dealt  with  this  subject 
agree  that  the  ash-content  of  etiolated  organs  is  relatively  greater  than 
in  green  stems.2 

There  is  not  general  agreement  as  to  the  distribution  of  nitrogen  in 
etiolated  plants,  but  there  seems  to  be  unanimity  in  the  conclusion 
that  the  total  nitrogen-content  is  less  than  in  normal  plants.  Accord- 
ing to  Karsten,  all  parts  of  the  plant  have  less  sugar  and  "gums"  when 
grown  in  darkness.3  As  the  gums  include  the  pentosans,  which  with  the 
nitrogenous  compounds  make  up  the  hydration  machine,  it  is  to  be 
seen  that  the  etiolated  plant  presents  the  features  of  lessened  protein, 
decreased  pentosans,  and  increased  salts,  all  of  which  would  tend  to  a 
lessened  imbibition  capacity  in  both  fresh  and  dried  material.  How 
much  the  lessened  nitrogen-content  would  contribute  to  this  general 
decrease  might  only  be  known  by  a  determination  of  the  character  of 
the  compounds  in  the  two  instances. 

All  of  the  facts  concerning  imbibition  by  living  plants  and  by  bio- 
colloids  tend  to  show  that  the  proposal  of  Palladin  to  ascribe  the  vari- 
ous departures  of  growth  in  plants  in  darkness  to  transpiration  effects 
is  not  tenable.  There  is  much  foundation  for  the  belief  that  form  may 
be  largely  affected  by  the  water-relation,  but  with  respect  chiefly  to 
imbibition  capacity,  as  a  resultant  of  the  protein-pentosan-salt  com- 
plex with  varying  acidity.  It  would  seem,  therefore,  that  for  Palladin's 
contention  that  the  ash  constituents  of  the  etiolated  plant  exercise  a  de- 
termining effect  on  the  growth  and  development  in  darkness  by  in- 
fluencing transpiration,  we  may  safely  substitute  an  assertion  that 
the  important  effect  of  the  salts  is  that  which  they  have  on  the  imbibi- 

1  MacDougal.     The  influence  of  etiolation  upon  chemical  composition,  in  "The  influence  of 
light  and  darkness  upon  growth  and  development."     Mem.  N.  Y.  Bot.  Garden,  2:  300-305. 
1891. 

2  Palladin,  W.     Eiweissgehalt  der  griinen  und  der  etiolirter  Blatter.     Ber.  d.  deut.  hot.  Ges., 
9:191.     1903. 

3  Karsten,  H.     Die  Einwirkung  dea  Lichtes  auf  das  Wachstums  der  Pflanzen.     Jena.     1870. 


Imbibition  and  Growth  of  Opuntia. 


135 


tion  or  absorption  action  of  the  plasmatic  colloids,  which  in  the  case 
of  Opuntia  are  probably  low  in  proteins.1 

The  growth  of  etiolated  shoots  of  Opuntia  is  of  an  indefinite  char- 
acter, as  the  length  which  such  members  may  reach  depends  to 
a  large  extent  upon  the  amount  of  available  material  and  other 
features.  Shoots  which  were  already  a  few  weeks  old,  and  which  had 
developed  in  a  dark  chamber  kept  at  16°  to  18°  C.  were  found  to  be 
growing  at  the  rate  of  5.2  mm.  daily.  These  were  removed  to  a 
second  chamber,  hi  which  the  temperature  was  kept  steadily  at  16°  C. 
for  3  days,  during  which  time  the  rate  varied  from  3.1  mm.  to  3.4  mm. 
daily.  The  temperature  was  brought  up  to  21°  to  23°  C.  in  3  hours, 
and  the  rate  was  5  mm.  for  the  first  day.  During  the  second  and 
third  days  at  this  temperature  the  rate  rose  to  7  mm.  per  day.  The 
rate  was  7.6  mm.  daily  during  the  next  2  days  and  about  8  mm.  daily 
for  a  final  period  of  16  hours.  The  temperature  now  being  raised  to 
30°  C.  in  2  hours,  and  after  that  varying  from  30°  to  32.5°  C.,  the  rate 
was  11  mm.  daily  during  the  first  16  hours,  then  at  the  rate  of  16.8  mm. 
during  the  succeeding  12  hours,  during  which  time  the  elongation  pro- 
gressed in  a  remarkably  uniform  manner. 


I2p.m.2    4     6     8     10  m.  24     6     6   10p.m. 


FIG.  24. — Auxographic  record  of  varia- 
tions of  length  of  etiolated  shoot  of 
Opuntia  X26,  at  temperatures  as  be- 
low, sheet  ruled  to  10  mm.  intervals:  (a) 
downward  movement  of  pen  7h30m  a.  m. 
to  9h40m  a.  m.  denoting  growth  at  tem- 
peratures of  the  stem  of  45°  to  49°  C.; 
(b)  growth  checked  for  20  minutes  at 
49°  C. ;  (c)  growth  resumed  at  temper- 
ature of  49°  C. ;  (d)  shortening  at  48.5° 
to  52°  C. ;  (e)  stationary  at  50.5°  C.; 
(/)  growing  at  temperatures  of  48°  to 
49°  C.;  (0)  shortening  at  49°  C.;  (h) 
growing  at  38°  to  41°  C. ;  (t)  shorten- 
ing at  49°  C. 


The  rate  itself  was  one  which  might  have  been  identified  with  that 
of  a  green  plant,  in  which,  however,  the  length  of  the  cell-mass  might 
not  be  equivalent.  The  chief  point  of  interest  in  the  present  con- 
nection is  that  which  comes  from  a  comparison  of  the  imbibition  capac- 
ity and  growth.  Sections  grown  at  17°  to  19°  C.  showed  an  imbibi- 
tion capacity  at  30°  to  31°  C.,  nearly  double  that  displayed  at  the  lower 
temperature,  and  it  was  also  to  be  seen  that  shoots  growing  at  the  rate 

x  Palladia,  W.     Transpiration  als  Ursache  der  Formanderung  etiolirter  Pflanzen.     Ber  d.  deut. 
bot.Ges.,  8:364.     1890. 


136 


Hydration  and  Growth. 


of  5.2  mm.  daily  at  16°  to  18°  C.  showed  a  rate  of  11  to  17  mm.  daily 
at  30°  to  32°  C.  The  rate  of  growth  would  be  one  which  would  be 
accepted  as  being  in  general  conformity  with  the  van't  Hoff  formula  of 
chemical  reaction,  while  as  a  matter  of  fact  it  is  not  widely  different  from 
the  capacity  for  imbibition  under  the  influence  of  equivalent  tempera- 
tures. Experimental  tests  have  already  been  described  in  which  the 
upper  limits  of  growth  and  the  behavior  of  etiolated  and  green  stems  of 
Opuntia  between  46°  and  51.5°  C.  are  not  widely  different.  (Fig.  24.)1 
The  rapid  and  wide  variations  at  these  high  critical  temperatures 
are  to  be  contrasted  with  the  steady  rate  of  a  growing  green  joint  main- 
tained at  30°  C.  for  38  hours.  The  illumination  in  the  daylight  period 
apparently  did  not  affect  the  rate  of  0.07  mm.  per  hour. 

12p.m.      m.         12p.m.      m         12p.m.     m.        12p.m.      m.       12  p.m. 


1 

1 

r  — 

\ 

1 

1 

I 

1 
1 

1 
I 

v  L 

\ 

• 

\ 

\  —  \  —  - 

t    '< 

1 

\          \ 

\      \ 

\      \ 

r^ 

\x*5\ 

\      \ 

\      \ 

\ 

\ 

\\ 

\       \ 

\ 

\ 

\ 

\ 

\ 

75 


FIG.  25. — Tracing  of  auxographic  record  of  variations  in  thickness  of  joint  of  Opuntia  approach- 
ing maturity.  Upward  movement  of  pen  denotes  increase  in  thickness,  X45.  Measurements 
made  from  place  between  areoles  near  base  of  joint.  Scale  ruled  to  5  mm.  and  12-hour 
intervals.  It  is  to  be  noted  that  the  hour  is  set  forward  on  the  summer  schedule. 

In  addition  to  the  great  number  of  records  of  the  variation  of  joints 
of  Opuntia  as  to  length,  a  few  days'  measurements  of  the  thickness  of  a 
maturing  joint  were  made  in  April  1918  hi  order  to  ascertain  whether 
or  not  increases  and  shrinkages  took  place  in  all  dimensions  at  the 
same  time.  The  auxographic  tracing  in  figure  25  gives  the  daily 
variation  in  thickness  near  the  base  of  a  maturing  joint,  which  are  seen 
to  be  correspondent  to  those  in  length.  It  is  to  be  noted  that  the 
instrument  was  necessarily  adjusted  so  that  increase  was  denoted  by 
the  upward  movement  of  the  pen,  in  a  manner  opposite  to  that  in 
nearly  all  the  other  records.  Later  the  bearing-lever  of  the  instru- 
ment was  adjusted  to  a  place  near  the  apex  of  the  joint  and  the  shrink- 
age, which  was  now  pronounced,  was  seen  to  set  hi  about  9  or  10  a.  m. 
and  continue  until  sunset,  at  which  time  thickening  began  and  lasted 
for  3  or  4  hours,  after  which  but  little  change  took  place  until  the  rising 
temperature  of  the  following  day  was  encountered. 

The  singular  retardations  and  variations  in  growth  which  are  highly 
characteristic  of  Opuntia,  together  with  its  unusual  features  of  trans- 
piration and  its  readily  measurable  imbibition,  offer  unusual  oppor- 
tunities for  the  examination  of  certain  agencies  affecting  growth, 
especially  as  the  action  of  some  of  these  factors  is  so  plainly  discernible 
in  the  variations  in  volume  of  mature  organs. 

1  MacDougal  and  Spoehr.     Growth  and  imbibition.     Proc.  Amer.  Phil.  Soc.,  56:  p.  308.     1917. 


Imbibition  and  Growth  of  Opuntia. 


137 


The  size  or  swelling  of  any  colloidal  mass,  such  as  a  growing  organ, 
or  particularly  a  joint  of  Opuntia,  will  depend  upon  the  integrated 
effects  of  several  agencies,  including  transpiration,  absorption,  the 
hydration  coefficient  (as  determined  by  the  acid,  salt,  and  protein 
content  of  the  protoplasm),  and  the  temperature. 

Several  series  of  measurements  of  the  separate  processes  involved 
have  been  made  at  the  Desert  Laboratory.  The  earliest  results  of 
importance  with  reference  to  transpiration  as  affecting  growth  were 
obtained  by  Mrs.  E.  B.  Shreve,  who  found  that  the  actual  amount  of 
water  which  an  excised  section  of  one  of  the  cylindropuntias,  Opuntia 
versicolor  (fig.  26),  might  contain  begins  to  decrease  some  time  after 
midday  and  continues  to  do  so  until  about  daybreak  of  the  fol- 
lowing morning,  then  increases  during  the  forenoon.1  If  the  plant 


B 


Apr.ZO 


Apr.ZZ         Apr.Z3          Apr.t4 


Fio.  26. 

A,  tracing  of  an  auxo- 
graphic  record  of  the 
variations  in  length  of 
a  stem  of  Opuntia  ver- 
sicolor. Below  is  a  line 
showing  the  daily  vari- 
ations in  temperature. 
Thegraph  is  composed 
of  sections  of  straight 
lines  representing  con- 
ditions of  illumina- 
tion. Interruptions 
of  the  sunlight  by 
cloudiness  are  shown 
on  April  21, 22, 23,  and 
29.  B,  auxographic 
record  of  variations 
in  thickness  of  stem 
of  Opuntia  versicolor, 
with  temperature  and 
illumination  indicat- 
ed. (After  Mrs.  E.  B. 
Shreve.) 

was  under  conditions  of  satisfied  imbibition  capacity,  the  plotted  line 
showing  such  capacity  would  also  be  that  of  growth.  Such  a  condition 
does  not  exist,  however.  Also,  if  the  simple  capacity  for  imbibition 
determined  volume,  and  this  capacity  were  always  satisfied,  the  plotted 
line  of  water  capacity  might  be  identical  with  that  of  the  daily  varia- 
tion in  volume  of  the  mature  organ.  Neither  does  this  condition 
exist  (fig.  27). 

The  record  of  variations  in  volume  of  a  joint  from  the  time  of  its 
beginning  to  maturity  and  through  the  following  season,  and  selected 
portions  of  this  graph,  are  reproduced  in  figures  28  and  29. 

The  aspect  of  the  daily  variations  in  volume  shows  seasonal  altera- 
tions and  depends  to  some  extent  upon  the  age  of  the  mature  joint, 
but  it  is  evident  that  the  joint  begins  to  increase  in  volume  in  the 

1  See  Shreve,  E.  B.     An  analysis  of  the  causes  of  variations  in  the  transpiring  power  of  cacti. 
Phyaiol.  Researches,  2:  No.  13.    August  1916. 


138 


Hydration  and  Growth. 


morning  or  at  some  time  in  the  forenoon  and  continues  to  do  so  until 
some  time  late  in  the  afternoon,  at  which  point  a  shrinkage  ensues  which 
continues  until  the  next  morning.  The  rate  of  water-loss  was  not  paral- 
lel with  the  changing  volume,  as  transpiration  was  most  rapid  between 
midnight  and  morning,  and  was  in  excess  of  the  amount  which  might 


Var. 


Tr. 


Ab. 


12p.m.  6a.m.     m.       6p.m    12p.m.     6a.m.      m.       6p.m. 

FIG.  27. — Absorption,  transpiration,  and  variation  in  length  of  growing  shoot  of  Opuntia.  Ab- 
sorption by  roots  denoted  by  dotted  line  is  least  about  midnight  and  is  greatest  about  midday, 
exceeding  transpiration  during  the  greater  part  of  the  daylight  period.  Transpiration  is 
greatest  about  midnight  and  least  about  midday,  according  to  data  by  Mrs.  E.  B.  Shreve. 
The  wavy  line  is  an  auxographic  tracing  of  actual  changes  in  length  of  a  joint  of  Opuntia 
in  a  mature  condition,  X  50. 

be  absorbed  by  the  roots  during  this  period.  It  is  obvious  without 
further  discussion  that  the  variation  in  volume  of  a  member  like  that 
of  a  joint  of  Opuntia  must  be  the  resultant  of  the  transpiration,  absorp- 
tion, and  water  capacity  of  the  cells  modified  by  the  action  of  the  new 
colloidal  material  which  may 

be  added  to  the  cell-masses.  TABLE  106. 

It  is  notable,  however,  that 
at  some  time  approaching 
midday  the  pen  recording 
the  variations  in  volume 
traces  a  line  not  far  from  the 
horizontal  for  a  brief  period, 
perhaps  half  an  hour,  at 
which  time  absorption  and 

transpiration  balance,  and  the  absolute  water  capacity  of  the  cell-masses 
is  greatest  (see  figs.  28  and  29).  An  equally  marked  confluence  of  fac- 


Time. 

Weight 

Increase 

Percentage 

in  grams. 

in  grams. 

increase. 

5h30m  a.  m.  .  . 

23.62 

6  30    a.  m.  .  . 

25.25 

1.63 

6.9 

4  30    p.  m.  .  . 

22.25 

5  30    p.  m.  .  . 

25.00 

2.75 

12.4 

Imbibition  and  Growth  of  Opuntia. 


139 


tors  is  to  be  seen  late  in  the  afternoon,  with  lessening  water  capacity, 
minimum  acidity,  and  the  beginning  of  decreased  absorption  by  the 
root-system. 

Some  measurements  of  growth  and  hydration  of  the  Platyopuntia, 
the  growth  of  which  has  been  observed  so  extensively,  were  made  by 
Mr.  E.  R.  Long  in  1915.  Imbibition  was  tested  by  noting  the  increases 


12p.m 


I?  p.m. 


12p.m. 


FIG.  28. — Portions  of  auxographic  record  of  variations  in  length  of  a  joint  of  Opuntia  from  October, 
1915  to  October  1916.  The  joint  had  formed  roots  which  depended  in  a  glass  jar  of  tap- water 
and  the  joint  was  held  firmly  in  a  natural  erect  position.  The  preparation  stood  in  the  south 
end  of  a  glass  house  and  was  exposed  to  the  alternations  of  sunlight  and  darkness  with  accom- 
panying temperatures  as  low  as  8°  C.  in  the  morning  and  as  high  as  45°  C.  in  midsummer 
afternoons.  The  actual  variations  are  amplified  30  and  elongation  is  denoted  by  the  down- 
ward movement  of  the  pen.  Divisions  of  scale  =1  cm.  The  joint  was  in  a  stage  of  com- 
pleted growth  during  the  week  beginning  October  18,  1915,  at  which  time  elongation  occurred 
from  evening  until  the  middle  of  the  forenoon,  and  shortening  during  the  remainder  of  the 
day,  with  the  length  less  at  the  end  of  the  week  than  at  the  beginning.  The  records  for  March 
and  April  show  a  similar  daily  periodicity,  but  with  an  increase  in  length  which  may  be 
ascribed  to  imbibition  under  the  advancing  temperatures.  The  daily  losses  and  gains  are 
more  nearly  equal  in  May  and  June  and  the  variations  of  narrow  amplitude.  Some  loss  is 
shown  in  July  and  the  reactions  of  the  joint  a  year  from  the  beginning  of  the  record  show  a 
daily  variation  different  in  many  particulars  from  those  of  the  previous  year.  See  figure  29 
for  a  continuation  of  the  record. 


140 


Hydration  and  Growth. 


TABLE  107. — Growth  of  flower-buds  of 
Opuntia,  March  25  to  April  24. 


in  weight  of  disk-shaped  sections  taken  from  the  joints  at  sunrise  and 
sunset,  with  the  results  shown  hi  table  106.     (Fig.  30.) 

These  results  afford  a  comparison  only  between  conditions  at  the 
beginning  and  end  of  the  daylight  period  and  the  tune  of  the  maximum 
and  minimum  imbibition  capacity  was  not  determined  as  in  Mesem- 
bryanthemum.  (See  p.  145).  Mr.  Long 
also  tested  the  effects  of  acids,  hy- 
droxids,  and  salts  upon  the  growth  of 
Opuntia.  Preparations  for  this  purpose 
consisted  of  mature  joints  bearing 
young  flower-buds.  The  bases  of  the 
joints  were  suspended  with  their 
freshly  cut  surfaces  in  solutions  in 
glass  jars,  and  the  lengths  of  the  buds 
were  taken  at  intervals  of  3  or  4  days. 
The  final  amount  of  growth  in  each 
case  is  given  in  table  107.  (Fig.  31. )* 


Total 

growth 

Medium. 

increment 

Time. 

before 

flowering. 

mm. 

days. 

Distilled  water  

42.0 

27 

N/50  NaOH  

40  5 

30 

N/50  malic  acid.  .  .  . 

36.0 

28 

N/50HC1  

31.0 

28 

12p.m.     m.       12p.m. 


12p.m. 


12p.m.      m.        12p.m.      m.       12p.m.      m.       12p.m. 


i 

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12  p.m. 


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DEC.  2&,  1916 

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1 

1 

1 

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APR.  2!?,  1917 

1 

!         1         '.    _ 

i 

•^     \                  ^-V  «v 

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FIG.  29. — Continuation  of  record  of  variations  in  length  of  mature  joint  of  Opuntia  (see  fig.  28) 
amplified  50  times.  The  amplitude  of  the  daily  variations  has  been  reduced  to  a  minimum 
November  to  January,  with  a  general  shrinkage  in  length.  Equable  conditions  accompanying 
clouds  and  rain  are  illustrated  by  the  record  of  the  week  beginning  January  15,  1917.  The 
influence  of  the  advancing  temperatures  in  inducing  increased  imbibition  is  illustrated  by 
the  records  of  February  to  April,  and  a  balance  of  daily  losses  and  gains  occurred  about  the 
end  of  April. 

1  Long,  E.  R.     Growth  and  colloid  hydratation  in  cacti.     Bot.  Gaz.,  59:  No.  6.     1915. 


Imbibition  and  Growth  of  Opuntia. 


141 


50 


o30 


£20 


10 


%o  Sodium  hydro/id 
Nutrient  solution 
Distilled  water 
%w  Hydrochloric  acid 
%o  Malic  acid 


%o  Hydrochloric  acid 


12 
Time  in  hours 


18 


Fia.  30. — The  course  and  amount  of  swelling  of  sections  of  Opuntia  in  solutions  as  indicated  dur- 
ing a  period  of  24  hours.  Fresh  sections  12  mm.  across  were  cut  from  green  joints  and  placed 
in  glass  dishes  in  a  dark  room  at  18°  C.  The  lines  are  traced  from  the  results  of  measure- 
ments made  at  6-hour  intervals.  Compare  with  fig.  31.  (Redrawn  after  E.  R.  Long.) 

50 


40 


30 


20 


10 


Nutrient  solution 
Distilled  water 

N/oo  Sodium  hydroxid 
N/so  Malic  acid 

%>o  Hydrochloric  acid 


April  10      13          16  20         23  27          30 

FIG.  31. — Record  of  growth  of  flower-buds  of  Opuntia,  on  joints  the  bases  of  which  were  immersed 
in  solutions,  the  imbibition  effects  of  which  are  shown  in  fig.  30.  The  record  covers  a  period 
of  three  weeks,  or  the  entire  measurable  period  of  extension  of  the  buds  to  the  time  of  open- 
ing. The  preparations  stood  in  a  glass  house  and  were  exposed  to  alternating  conditions  of 
daylight  and  darkness  and  to  temperatlres  of  about  15°  to  30°  C.  (See  fig.  30.)  (Redrawn 
after  E.  R.  Long.) 


142 


Hydration  and  Growth. 


Mr.  Long's  results  were  confirmed  by  the  author  in  April  1918,  at 
which  time  auxographic  methods  and  technique  of  measuring  the 
swelling  of  such  sections  had  been  brought  to  an  advanced  stage  of 
efficacy.  Sections  were  cut  at  sunrise,  noon,  and  sunset,  from  young 
joints  8  to  10  cm.  long  growing  hi  the  open  under  natural  conditions. 
Such  sections  had  an  average  thickness  of  about  4  mm.  and  when 
swelled  at  20°  C.  gave  the  increases  shown  in  table  108. 

TABLE  108. 


Opuntia, 
median 
sections. 

Time  taken. 

Dried 
slice 
taken 
7  a.  m. 

7  a.  m. 

8  a.  m. 

Noon. 

5  p.  m. 

Distilled  water.  .  .  . 
Citric  acid  

p.  ct. 
7 
6 

p.  ct. 
4.6 
6 
8.2 

p.  ct. 
8.2 
8.2 
12.0 

p.  ct. 
4.6 
5.2 
11.8 

392 
400 
440 

Sodium  hydroxid.  . 

The  great  increase  of  dried  slices  is  indicative  of  high  water  capacity 
of  living  material.  The  relative  swelling  of  the  sections  in  the  different 
solutions  is  identical  with  that  of  the  fresh  sections,  demonstrating  that 
the  dominant  process  is  imbibition  rather  than  osmosis. 

The  time  required  for  satisfaction  varied  widely  with  the  time  at 
which  material  was  taken  and  the  character  of  the  solution.  The 
sections  taken  at  the  end  of  the  day  were  fully  hydrated  in  distilled 
water  and  began  to  shrink  in  6  hours.  The  sections  taken  at  sunrise, 
which  were  most  highly  acid,  as  those  taken  in  the  evening  are  least 
acidified,  were  satisfied  in  2  hours  and  began  to  shrink  in  3  hours.  The 
material  taken  in  the  morning  was  saturated  in  the  acid  solution  in  less 
than  an  hour  and  was  shrinking  rapidly  an  hour  and  a  half  after 


8a.m. 


8  a.m. 


I    I    I    I    I 


FIG.  32. — The  tracing  on  the  left  shows  the  variation  in  volume  of  a  trio  of  sections  of  a  young 
joint  of  Opuntia  4.6  mm.  in  thickness  which  swelled  6.5  per  cent  in  citric  acid,  0.01  N,  less 
than  three  hours  at  20°  C.,  then  began  to  shrink.  The  tracing  on  the  right  shows  the 
reaction  of  a  similar  trio  which  had  an  average  thickness  of  but  3.9  mm.,  and  which 
swelled  6.4  per  cent  in  an  hour  at  28°  C.  and  then  began  to  shrink  very  rapidly. 

immersion  (see  fig.  32).  Swelling  in  the  alkaline  solution  was  charac- 
teristically slow  and  long-continued.  Material  taken  in  the  morning 
continued  to  increase  for  4  hours,  and  that  taken  hi  the  evening  was 
still  showing  some  increase  12  hours  later. 

Another  feature  in  conformity  with  the  condition  of  the  joint  at  differ- 
ent times  of  the  day  was  the  fact,  confirmed  by  repeated  tests,  that  the 


Imbibition  and  Growth  of  Opuntia.  143 

difference  between  the  amount  of  swelling  of  sections  in  distilled  water 
taken  at  sunrise  and  swelled  at  20°  and  28°  C.  was  so  small  as  to  be 
negligible.  The  same  condition  prevailed  in  dried  slices  taken  in  the 
morning  and  dried  rapidly  in  the  desiccator.  Slices  which  came  down 
to  a  thickness  of  about  0.6  mm.  swelled  392  per  cent  at  20°  C.  and  a 
second  set  increased  an  equivalent  proportion  at  27°  to  28°  C.  The 
sets  of  living  sections  used  for  these  tests  gave  an  increase  of  about  6.5 
per  cent  in  water.  Sections  taken  at  noon  swelled  8.2  per  cent  at  20°  C. 
and  9.8  per  cent  at  28°  C.  Sections  taken  at  the  end  of  the  day 
swelled  4.6  per  cent  at  20°  C.  and  6.5  per  cent  at  28°  C.  The  material 
used  for  these  paired  tests  was  selected  to  be  equivalent  as  far  as  pos- 
sible, and  comparisons  transverse  to  those  stated  are  not  allowable. 

The  acidosis  produced  by  the  residual  acids  of  the  morning  condition 
may  be  taken  to  be  accentuated  by  the  rising  temperature  and  to 
cancel  or  mask  any  additional  absorption  which  might  accrue  as  a 
direct  effect  of  temperature  on  a  neutral  solution.  The  increases  at 
other  times  would  be  due  to  the  direct  action  of  rising  temperatures 
on  absorption. 

It  became  evident  in  the  earliest  work  done  with  the  opuntias  that 
the  young  joints  showed  a  swelling  in  hydroxid  solutions  greater  than 
in  water  or  acids,  a  fact  probably  attributable  to  the  formation  of 
compounds  of  the  sodium  hydroxid  with  the  carbohydrates  present, 
in  conjunction  with  possible  sodium  albuminates.  The  carbohydrates 
are  in  greatest  proportion  in  young  joints.  The  swelling  in  hydroxid 
in  later  stages  comes  down  to  a  level  near  that  in  acid  and  in  water. 
An  additional  fact  of  interest  was  the  action  of  the  juice  of  the  joints 
taken  in  the  condition  in  which  it  is  found  at  midday.  Duplicates  of 
the  dried  slices,  which  have  already  been  noted  as  showing  an  increase 
of  390  per  cent  in  distilled  water,  swelled  325  per  cent  at  20°  C.  in  the 
expressed  juice.  Similar  dried  slices  swelled  372  per  cent  in  the  freshly 
expressed  juice  of  Echinocactus  wislizeni.  Its  hydrating  effect  on  thin 
plates  of  a  biocolloid  consisting  of  agar  6  parts,  opuntia  mucilage  2 
parts,  bean  protein  1  part,  and  gelatine  1  part  was  much  marked. 
Such  sections  swelled  2,450  per  cent  at  20°  C.  in  distilled  water,  and 
only  700  per  cent  in  the  fresh  juice  of  Opuntia  taken  at  midday. 

The  principal  factors  which  influence  the  rate  and  course  of  growth 
of  Opuntia  have  been  described  in  the  preceding  pages.  The  joints 
when  in  the  youngest  stage  have  a  mode  of  growth  which  does  not 
differ  materially  in  the  record  which  it  makes  from  that  of  many  other 
green  plants.  As  soon  as  it  reaches  medium  size  its  innate  peculiarities 
of  transpiration  and  metabolism  and  certain  morphological  features 
operate  to  give  it  a  highly  characteristic  daily  chart  of  elongation  and 
retardation  or  shrinkage.  The  respiration  of  these  plants  is  of  a 
character  which  results  hi  the  accumulation  of  acids  to  an  amount 
equivalent  to  as  much  as  0.1  N  malic  acid  at  daybreak,  which  is  suffi- 


144  Hydration  and  Growth. 

cient  to  have  a  distinct  effect  on  the  hydration  capacity  of  the  cell- 
colloids.  Late  in  the  daylight  period  the  acid  may  be  reduced  to  a 
point  below  the  hundredth  normal  which  has  been  used  so  exten- 
sively in  this  work  as  a  standard  solution.  The  effects  of  acidosis 
would,  of  course,  vary  as  the  composition  of  the  pentosan-protein-salt 
colloid  of  the  joint  passed  from  its  embryonic  aspect  to  that  of  the 
mature  member.  The  actual  amount  of  water  lost  by  the  greater 
number  of  plants,  especially  those  with  thin  stems  and  broad  leaves, 
is  greater  for  the  daylight  period  than  for  the  night.  Opuntia  is  a 
notable  exception  to  this  generalization,  and  its  rate  of  transpiration 
is  greatest  during  the  night,  usually  between  midnight  and  morning. 
All  of  these  agencies  affecting  imbibition  likewise  have  a  determining 
influence  oh  growth,  and  the  resultants  in  Opuntia  and  presumably  in 
other  massive  succulents  are  such  as  to  constitute  a  characteristic 
type  of  growth. 

Another  feature  about  which  but  little  has  been  said  is  that  of  the 
morphology  of  the  compound  members  of  the  stems  of  Opuntia.  At 
first,  when  a  young  joint  is  but  2  or  3  cm.  in  length,  it  is  wholly  in  an 
embryonic  condition  and  its  colloids  show  the  reactions  of  such  mix- 
tures. As  development  progresses  much  permanent  tissue  is  formed, 
hi  which  finally  the  embryonic  tracts  lie  as  a  network  or  reticulum. 
Any  given  section  of  a  growing  joint  contains  growing  and  mature 
tissue  after  a  certain  stage  is  reached,  but  when  the  mature  tissue 
reaches  a  proportion  something  greater  than  that  of  the  growing 
masses  its  characteristic  variations  in  hydration  overshadow  those  of 
the  growing  cells  and  give  rise  to  the  retardations  and  shrinkages 
which  are  so  marked  a  feature  of  these  plants.  It  is  probable  that 
many  features  of  the  rate  and  course  of  growth  may  be  ascribed  to 
anatomical  relations,  of  which  the  above  is  an  illustration. 


XL  THE  HYDRATION  REACTIONS  AND  GROWTH  OF  MESEM- 
BRYANTHEMUM,  HELIANTHUS,  AND  PHASEOLUS. 

The  results  obtained  by  a  study  of  the  hydration  of  the  cacti  are 
especially  valuable  because  of  the  possibility  of  their  correlation  with 
features  of  varying  composition  which  could  be  determined  by  chemical 
analyses.  Measurements  of  a  second  type  of  succulent  were  sought 
for  the  purpose  of  bringing  into  relief  the  possibilities  of  rapid  changes 
in  the  water-content  of  growing  and  mature  organs.  A  mesembryan- 
themum  (Mesembryanthemum  edule)  which  flourishes  in  the  open 
at  the  Coastal  Laboratory  and  in  the  glass-house  at  the  Desert  Labora- 
tory furnished  material  suitable  for  such  studies.  The  leaves  attain  a 
length  when  mature  of  about  6  to  10  cm.  and  are  triangular  in  cross- 
section,  the  three  faces  being  about  10  to  12  mm.  across.  Metabolism 
runs  a  course  in  these  organs  similar  to  that  of  the  opuntias,  as  a  result 
of  which  acid  accumulates  during  the  night,  and  decreases  with  the 
disintegrating  action  of  light  during  the  daytime,  as  illustrated  by  the 
data  given  in  table  109.  The  total  daily  range  in  the  concentration  of 
the  acids  is  much  less  than  that  displayed  by  the  cacti. 

TABLE  108. — Acidity  of  juice  of  leaves  of  Mesembryanthemum  in  cubic  centimeters 

of  N/100  KOH. 


8  a.  m. 

Noon. 

4h30m  p.  m. 

Sample  A. 
Fresh  juice,  per  c.c  

p.  ct. 
0.0280 

p.  ct. 
0.0279 

p.  ct. 
0.0232 

Total  acidity  per  gm.  dry  material  .  . 
Total  acidity  per  gm.  fresh  material  . 

Sample  B. 
Pure  juice,  per  c.c  

1.584 
.0356 

.0273 

1.509 
.0351 

.0225 

1.191 
.0264 

.0205 

Total  acidity  per  gm.  dry  material  .  . 
Total  acidity  per  gm.  fresh  material  . 

1.072 
.029 

1.091 
.0241 

1.056 
.0275 

Measurements  of  the  varying  diameter  of  young  and  of  mature 
leaves  indicate  that  the  direct  water-loss  from  the  surfaces  are  so  im- 
portant as  to  mask  the  imbibition  capacity  as  affected  by  acidity  and 
other  factors,  as  will  be  apparent  from  auxographic  measurements. 

Mention  has  been  made  previously  of  the  general  similarity  of  the 
course  of  growth  of  Mesembryanthemum  to  that  of  Opuntia.  It  was 
possible  to  go  into  this  matter  in  greater  detail  in  the  experiments 
described  in  this  volume.1 

A  series  of  tests  was  arranged  to  ascertain  the  alterations  in  volume 
of  these  leaves  both  in  a  mature  condition  and  when  in  the  course  of 


1  MacDougal  and  Spoehr.     Growth  and  imbibition.     Proc.  Amer.  Phil.  Soc.,  56:  314.     1917. 

145 


146 


Hydration  and  Growth. 


growth.  Their  regular  surfaces  made  it  possible  to  place  one  side 
snugly  on  a  small  wooden  block,  and  to  bring  the  cork-tipped  vertical 
auxograph  lever  in  bearing  with  the  uppermost  angle  of  the  leaf.  The 
stem  was  held  firmly  a  few  centimeters  from  the  base  of  the  leaf,  which 
was  exposed  to  no  disturbing  conditions  (fig.  33).  First,  the  diurnal 
variations  of  a  mature  leaf  were 
followed  without  any  attempt  being 
made  to  equalize  temperatures, 
which  were  taken  by  a  thermometer 
with  a  thin  bulb  thrust  into  a 
second  leaf  and  allowed  to  remain 
there.  The  readings  were  as  low 
as  10°  C.  at  daybreak  and  as  high 
as  31°  C.  at  midday.  The  varia- 
tions in  volume  amplified  45  times 
shown  hi  figure  34.  The  record 
beginning  at  noon  on  February  14, 
with  the  temperature  of  the  leaves 
at  25°  C.,  was  at  a  juncture  when 
shrinkage  set  in,  lessening  the 
thickness  of  the  leaf  about  1  mm., 
or  10  per  cent  of  its  turgid  thick- 
ness before  5h30m  p.  m.,  at  which 
time,  with  the  temperature  still  at  a 
high  point  (27°  C.),  the  shrinkage 
came  to  an  end  and  enlargement 
began,  which  continued  through  the 
night,  so  that  at  8  o'clock  the  follow- 
ing morning  the-  leaf  was  actually 
thicker  than  on  the  preceding  day. 
The  temperature  on  this  day  rose 
to  31°  C.  and  the  shrinkage  exceeded 
that  of  the  preceding  day,  amount- 
ing to  about  13  per  cent  of  the 
turgid  thickness  of  the  leaf.  Swell- 
ing began  again  in  the  evening, 
which  restored  the  leaf  to  about  its 
original  dimensions  48  hours  after 
the  beginning  of  the  record. 

The  variations  appeared  to  run  parallel  in  part  only  to  those  already 
described  in  detail  f  or  Opuntia,  and  to  the  extent  that  the  daily  variations 
take  the  form  of  alternate  shrinking  and  enlargement  with  the  increase 
in  excess  of  the  loss.  The  chief  features  of  growth  may  be  illustrated  by 
the  record  of  one  of  a  pair  of  leaves  which  had  attained  about  two-thirds 
of  the  full  size.  This  was  put  in  bearing  with  the  auxograph  in  the 


FIG.  33. — Detail  of  arrangement  of  auxo- 
graph to  record  variations  in  thickness  of 
leaf  of  Mesembryanthemum.  Leaf  in  a 
horizontal  position  resting  on  a  wooden 
support  cut  away  below  to  make  place  for 
the  younger  terminal  leaves. 


Some  Hydration  Reactions  and  Growth. 


147 


I2p.m.       m.        J2p.m. 


T 


X45 


12  p.m. 


manner  described,  and  a  portion  of  the  record  is  given  in  figure  35.  As 
may  be  seen,  the  period  of  shrinkage,  which  begins  about  the  same  time 
in  the  older  leaf,  continues  for  a  shorter  period,  on  some  days  not  more 
than  2  hours,  and  enlargement  sets  in  in  mid- 
afternoon.  The  thickness  on  each  successive 
morning  was  greater  than  at  the  same  tune  on 
the  preceding  day,  demonstrating  that  actual 
growth  was  in  progress. 

Two  series  of  measurements  were  now  un- 
dertaken to  secure  new  records  of  the  elonga- 
tion of  leaves  which  had  reached  about  half 
the  final  length  and  of  others  still  younger. 
Such  a  pair  of  young  leaves,  with  their  surf  aces 
still  appressed  in  an  erect  position,  were 
brought  into  bearing  on  an  auxograph  lever  in 
a  sunny  place  in  a  glass-house.  The  length  of 
the  exposed  portion  was  about  25  mm.  and 
their  thickness  was  not  over  0.5  mm.  at  the 
beginning  of  the  tests.  Here,  as  in  previous 
preparations,  it  was  found  that  whatever  the 
causes  of  the  stoppage  of  growth  and  of 
shrinkage  might  be,  they  were  not  effective  in 
producing  an  actual  cessation  of  elongation, 
which  hi  these  young  leaves  continued  through- 
out the  24  hours  of  the  day  variously  respon- 
sive to  alterations  in  temperature  (fig.  36). l 

Another  pair  which  were  about  to  spread  by  the  growth  of  the  bud 
ensheathed  between  their  bases  were  attached  to  the  auxograph  and 
set  in  a  place  where  they  would  be  shielded  from  the  direct  rays  of  the 
sun.  Preparations  of  all  three  stages  increased  in  length  and  thickness 


FIG.  34. — Upper  part  of  fig- 
ure is  an  auxographic  rec- 
ord of  variations  in  thick- 
ness of  leaf  of  Mesembry- 
anthemum  on  a  cloudy 
day  with  but  little  change 
in  temperature,  as  taken 
by  a  mercurial  thermom- 
eter from  a  similar  leaf. 
Upward  course  of  line  de- 
notes increase.  X45. 

Lower  figure  is  an  auxo- 
graphic tracing  of  same 
leaf  on  sunny  day  with 
temperatures  of  16°  to  25° 
C.  Shrinkage  occurred 
during  the  entire  day- 
light period.  X45. 


5 
15 
25 

35 
45 
55 
65 


I2p.m        m. 


12p.m.       m.         12p.m.      m.         12  p.m.       m. 


T 


/ 


_T 


I 


24"C, 


,27'C. 


\ 


\ 


\ 


± 


FIG.  35  — Auxographic  record  of  thickness  of  pair  of  leaves  about  half  mature  size.  X45.  A 
daily  shrinkage  between  8  a.  m.  and  midday  occurred,  which  amounted  to  an  increasing  pro- 
portion of  the  increase  taking  place  during  the  remainder  of  the  day. 

during  the  entire  night,  the  increase  in  thickness  being  very  rapid 
during  the  first  half  of  the  night  and  slowing  down  to  a  very  low  rate 

1MacDougal  and  Spoehr.     Growth  and  imbibition.     Proc.  Amer.  Phil.  Soc.,  56:  310.     1917. 


148 


Hydration  and  Growth. 


afterward,  beginning  actual  shrinkage  by  9  a.  m.  Growth  of  the  leaf 
of  middle  age  slows  down  with  the  low  temperature  of  daybreak,  but 
accelerates  at  9  a.  m.,  at  the  time  the  older  leaf  begins  shrinking,  at 
17°  C.  The  youngest  pair  of  leaves  grows  with  a  high  rate  all  night, 
with  a  perceptible  slowing  at  daybreak,  but  it  also  accelerates  at  9  a.  m. 
with  the  rising  temperature,  at  the  time  the  oldest  leaves  are  shrinking 
(fig.  37). 


12p.m.        m. 


12p.m. 


15 
25 
35 
45 
55 
rr 

V      /      / 

\ 

I4*C.\ 

zz'o. 

\ 

\ 

X20 

\  v° 

f- 

12  p.m. 


12p.m. 


5 
15 
25 
35 
45 
55 

'               /               / 

J        /        / 

[\I6°C. 

J 

[ 

I8°CV 

otfr 

X20 

^ 

"X 

i 

i 

\ 

B 


I2p.m. 


3l°C. 


FIG.  36. — A,  auxographic  record  of  elongation  of  pair  of  leaves  2  cm.  long  during  a  24-hour 
period;  temperatures  of  14°  to  25°  C.  Retardation  during  period  of  highest  temperature  is 
illustrated.  X20.  B,  action  of  same  leaves  in  shade. 

The  general  features  of  growth  under  the  usual  varying  conditions 
of  alternating  daylight-high  temperature  and  night-low  temperature 
complexes  being  determined,  it  became  necessary  to  test  the  swelling 
of  the  leaves,  as  had  been  done  with  the  joints  of  Opuntia  to  ascertain 
their  unsatisfied  hydration  capacity. 

Preparations  for  testing  the  swell- 
ing of  living  material  of  leaves  were  s 
made  by  placing  these  triangular  !5 
organs  on  a  flat  surface  alongside  a  25 
guide  of  5  mm.  in  thickness.  A 
razor  slid  along  this  slices  away  the 
uppermost  angle,  leaving  a  trun- 
cated section  5  mm.  in  thickness,  in 
which  the  central  fibrovascular 
tissue  could  be  seen  through  the 
translucent  parenchymatous  tissue. 
Segments  about  1  cm.  long  were 
taken,  to  the  exclusion  of  the  basal  and  apical  parts  of  the  leaf.  It  is 
to  be  noted  that  active  enlargement  is  usually  in  progress  morning 
and  evening  in  young  leaves,  while  mature  leaves  are  enlarging  in  the 
morning  but  shrinking  in  the  afternoon  and  night.  The  results  of  the 
hydrations  are  shown  in  table  110. 

In  the  above  tests  the  amount  of  swelling  was  most  in  distilled  water, 
less  in  hydroxid,  and  least  in  acid,  in  mature  leaves  taken  in  the  even- 


35 


45 


55 


FIG.  37. — Variations  in  thickness  of  a  mature 
leaf  of  Mesembryanthemum.  X45.  Tem- 
peratures taken  from  a  similar  leaf  with 
a  mercurial  thermometer. 


Some  Hydration  Reactions  and  Growth. 


149 


ing  while  in  a  shrinking  stage.    These  differences  hold  for  the  morning, 
except  that  the  effects  of  acid  and  hydroxid  are  about  equal. 


TABLE  110. 


Mesembryanthemum, 
median  slices  of 
leaves. 

Taken  at  6  p.  m., 
swelled  at 
15  to  17°  C. 

Taken  at  8  a.  m., 
swelled  at 
13  to  15°  C. 

Mature 
leaves. 

Young 
leaves. 

Mature 
leaves. 

Young 
leaves. 

Distilled  water  

p.  ct. 
18 
16 
15 

p.  ct. 
12 
16 
12 

p.  ct. 
13 

8 
8 

p.  ct. 
22 
13 
47 

Sodium  hydroxid,  0.01  M 
Citric  acid,  0.01  N  

An  additional  set  of  measurements  with  young  leaves  decreased  the 
differential  between  the  more  acid  condition  of  the  morning  and  the 
less  acid  condition  of  the  evening,  as  is  shown  in  table  111. 


TABLE  111. 


Young  leaves, 

Young  leaves, 

taken  at  6 

taken  at  8 

Mesembryanthemum. 

p.   m.,   and 
swelled     in 

a.  m.,   and 
swelled     in 

darkness  at 

darkness  at 

12  to  15°  C. 

12  to  15°  C. 

p.  ct. 

p.  ct. 

Distilled  water  

13 

12 

Sodium  hydroxid,  0.01  M  .  . 

11 

10 

Citric  acid,  0.01  N  

11 

10 

Young  leaves  are  generally  in  a  state  of  enlargement  both  morning 
and  evening,  and  should  show  but  little  difference  in  swelling  capacity 
in  the  two  stages,  while  mature  leaves  are  enlarging  in  the  morning 
and  shrinking  hi  the  afternoon,  in  expression  of  a  condition  which  is 
reflected  in  the  swelling  reactions.  Thus,  for  example,  sections  of 
mature  leaves  taken  in  the  morning,  which  had  a  thickness  of  about 
9.3  mm.,  swelled  6  per  cent  at  14°  to  19°  C.  in  the  dark  room,  which  was 
about  the  temperature  at  which  they  were  taken.  An  equivalent  set 
taken  at  1  p.  m.  swelled  15  per  cent  in  water  at  17°  to  19°  C.  From 
which  it  may  be  seen  that  the  measurements  of  variations  in  thickness 
of  entire  leaves  (see  pp.  147,  148)  are  in  entire  conformity  with  other 
known  facts. 

The  foregoing  measurements  were  obtained  from  segments  cut 
from  leaves  about  1  cm.  in  length  and  with  the  epidermis  on  the  three 
faces  intact.  The  measurements  made  for  the  purpose  of  determining 
the  possible  effects  of  acidity  were  obtained  by  cutting  slices  which 
removed  one  angle  of  the  leaf  and  the  epidermal  face  parallel  to  the 
excised  surface.  If  the  water-loss  is  a  factor,  its  effects  would  be  most 


150  Hydration  and  Growth. 

pronounced  in  the  layers  which  had  been  removed.  The  mature 
leaves  were  distinctly  flabby  to  the  touch  and  the  external  layers  were 
in  a  state  of  partial  collapse  during  the  midday  period. 

Segments  of  young  leaves  in  the  stage  in  which  these  organs  con- 
tinued elongation  and  increase  during  the  entire  day  were  now  taken 
for  comparison.  Their  thickness  was  a  little  more  than  half  that  of 
the  mature  leaves.  The  trio  of  sections  taken  from  leaves  at  8  a.  m., 
when  the  highest  turgidity  prevailed,  showed  a  swelling  of  13.6  per  cent, 
while  those  taken  at  midday  swelled  16.7  per  cent  at  14°  to  20°  C. 
The  difference  in  the  two  cases  is  much  less  proportionately  than  that 
which  is  set  up  in  mature  leaves  and  fully  accounts  for  the  shrinkage 
of  the  older  organs.  Beyond  this  the  auxographic  tracings  made  obvi- 
ous another  difference  between  the  young  and  mature  leaves.  The 
total  swelling  in  the  mature  leaves  was  reached  in  6  or  8  hours,  most 
of  which  developed  within  2  hours  of  immersion.  The  swelling  of 
young  leaves  was  much  more  gradual,  and  the  rate  was  less  rapid  at 
first  and  then  decreased  much  more  gradually  and  had  not  actually 
ceased  at  the  end  of  20  hours.  The  material  in  the  young  leaves  was 
not  only  alive,  but  in  a  growing  condition;  consequently  new  colloidal 
material  in  the  process  of  aggregation  would  provide  a  continuing 
source  of  hydration  capacity. 

Confirmatory  evidence  consists  in  the  fact  that  when  two  pairs  of 
leaves  are  put  in  bearing  with  auxographs,  the  one  exposed  to  the  sun 
soon  reaches  the  stage  where  the  water-loss  at  midday  is  equivalent 
to  growth  and  neutralizes  it  on  the  record,  while  the  pair  of  leaves 
of  the  same  age  shaded  from  the  sun  continues  elongation  scarcely 
checked  or  retarded  during  the  same  period.  Now,  if  the  preparation 
in  the  sunny  location  experiences  a  cloudy  day  or  is  shaded,  it  too 
continues  growth  during  the  entire  day  in  a  manner  which  shows  con- 
clusively that  the  daily  retardation  is  in  the  main  due  to  excessive 
water-loss  in  the  case  of  the  Mesembryanthemum.  A  similar  action  by 
the  cacti  has  already  been  discussed  in  Chapter  X. 

The  practice  of  testing  the  swelling  of  dried  sections  for  comparison 
with  that  of  living  material  was  followed  as  in  Opuntia.  Sections  of 
living  leaves  about  5  mm.  in  thickness  were  prepared  as  above,  and 
these  were  placed  between  folds  of  filter-paper  and  weighted  only 
sufficiently  to  prevent  warping  and  curling  during  desiccation,  which 
continued  for  a  week.  Their  final  thickness  was  about  0.25  mm.  and 
their  swelling,  unlike  that  of  the  segments  of  Opuntia,  did  not  come 
back  to  the  approximate  size  of  the  fresh  material.  The  final  measure- 
ments in  percentages  of  the  dry  thickness  were  as  follows: 

TABLE  112. 

p.  ct. 

Distilled  water 100 

Citric  acid,  0.01  N 120 

Sodium  hydroxid,  0.01  M 100 


Some  Hydration  Reactions  and  Growth.  151 

It  had  been  previously  concluded  that  the  dominant  factor  in  the 
varying  rate,  and  in  producing  shrinkage,  was  that  of  modified  hydra- 
tion  capacity  as  dependent  chiefly  upon  the  acidity  of  the  sap  and  the 
balance  between  absorption  and  water-loss  by  transpiration.  The 
effects  of  the  last-named  feature  were  marked,  as  the  experiments  were 
carried  out  under  conditions  of  drought  approaching  the  limit  of 
endurance  of  this  plant.  Sudden  changes  in  hydrogen-ion  concen- 
tration of  the  sap  of  another  species  of  this  genus,  with  other  unusual 
features  of  the  sap  as  noted  by  J.  Hempel,  may  be  responsible  for  some 
of  the  aberrances  shown  by  this  plant.1  While  this  author  gives  nitro- 
gen determinations  showing  much  greater  uniformity  than  in  other 
succulents,  this  uniformity  of  total  nitrogen  may  include  many  changes 
in  amino-groups  which  might  affect  the  water  capacity  of  the  colloids. 
One  species  was  found  especially  high,  in  nitrogen. 

The  sunflower  has  been  used  extensively  in  studies  on  growth- 
rates,  and  the  behavior  of  young  and  older  internodes  of  the  same  stem 
of  Helianthus  furnishes  some  homologies  with  the  alterations  exhibited 
by  the  succulents.  A  stock  of  Helianthus  annuus  was  grown  in  the 
glass-house  of  the  Desert  Laboratory  in  February  and  March  1918, 
at  which  time  they  showed  vigorous  and  normal  development.  Dur- 
ing most  of  the  time  in  which  these  observations  were  made  the  tem- 
perature of  the  air  rose  to  25°  to  31°  C.  in  the  glass-house  and  some 
slight  wilting  effects  were  noticeable  in  the  leaves  at  midday,  a  fact 
included  in  the  records  given  below,  to  which  are  also  attached  the  tem- 
peratures taken  by  thermometers  thrust  into  the  stems.  The  young 
parts  in  which  growing  cells  constitute  the  greater  part  of  the  mass  con- 
tinue to  increase  during  a  period  in  which  the  older  parts  are  shrinking. 
The  older  parts  consist  of  cylinders  of  tissue,  almost  mature,  fully 
saturated,  with  no  continuing  increase  in  hydration  capacity;  and  the 
growing  cells  are  in  the  form  of  an  irregular  cylindrical  shell  under- 
neath the  epidermis,  the  thickness  of  which  is  no  more  than  a  small 
fraction  of  the  entire  diameter.  Consequently  the  external  measure- 
ments of  the  stem  are  chiefly  determined  by  the  changes  in  the  mature 
cell-masses. 

The  preliminary  swelling  tests  were  made  with  sections  of  the  ter- 
minal internodes  from  which  tangential  slices  had  been  removed,  leav- 
ing them  with  a  thickness  of  2.7  mm.  Such  sections  at  14°  to  16°  C. 
swelled  7.4  per  cent  in  distilled  water,  9.2  per  cent  in  hundredth-normal 
sodium  hydrate,  5.5  per  cent  in  a  similar  solution  of  citric  acid,  and 
7.4  per  cent  in  a  similar  solution  of  potassium  nitrate.  These  results 
are  fairly  representative  of  this  type  of  plants. 

The  preceding  series  was  made  up  before  the  daily  shortage  of  water 
in  the  terminal  parts  of  the  stems  had  been  detected.  A  second  set  of 

1  Hempel,  J.     Buffer  processes  in  the  metabolism  of  succulent  plants.      Compt.  Rend.  d.  Trav. 
d.  Lab.  d.  Carlsberg,  13.     1917.     See  pp.  45-51. 


152  Hydration  and  Growth. 

sections  were  therefore  taken  at  Ih30m  p.  m.,  when  the  plant  stood  at  a 
temperature  of  22°  C.,  and  these  were  swelled  hi  the  dark  room  at 
once  with  solutions  which  stood  at  18°  to  20°  C.  during  the  time  of  the 
swelling.  The  increases  were  as  follows: 

TABLE  113. 

p.  ct. 

Distilled  water 70 

Citric  acid,  0.01  N 19 

Sodium  hydroxid,  0.01  M 46 

Potassium  chloride,  hydrochloric  acid,  0.01  M 14 

The  examination  of  the  sections  after  the  records  were  complete 
showed  that  they  were  variously  twisted  and  curled,  due  to  the  fact 
that  the  internal  parenchymatous  tissues  had  swelled  more  than  the 
external  layers.  So  far  as  could  be  estimated  by  simple  observation 
without  measurement,  the  error  did  not  double  the  measurement,  how- 
ever. Consequently  it  is  to  be  seen  that  the  imbibition  capacity  of 
these  stems,  due  to  a  depletion  of  the  water-balance,  is  much  greater  at 
noon  than  in  early  morning. 

A  repetition  of  the  first  test  with  whole  sections  that  could  not  so 
readily  twist  showed  that  another  series  of  sections  taken  at  8  a.  m. 
swelled  as  follows,  at  18°  to  20°  C.: 

TABLE  114. 

p.  ct. 

Distilled  water 4 

Citric  acid,  0.01  N 4 

Sodium  hydroxid,  0.01  M 6.5 

Potassium  chloride,  hydrochloric  acid,  0.01  M 6 

This  series  was  characterized  by  the  satisfaction  of  the  full  hydration 
capacity  within  an  hour  or  two,  except  in  the  case  of  the  alkaline  solu- 
tion, in  which  the  increase  was  very  gradual.  The  material  had  re- 
turned to  its  original  dimensions  within  6  hours  in  the  other  liquids 
and  continued  to  shrink.  This  action,  coupled  with  decoloration,  was 
especially  marked  in  the  acidified  saline  solution.  As  a  still  further 
verification  of  the  above  results,  a  trio  of  sections  exactly  like  those  of 
the  above  series  were  taken  at  midday  on  the  following  day,  and  these 
swelled  14  per  cent  in  water,  while  another  trio  increased  10  per  cent  in 
the  acidified  saline  solution  before  shrinking. 

The  earlier  measurements  of  the  swelling  of  the  sunflower  having 
been  made  with  the  terminal  internodes  of  growing  stems,  a  final  series 
was  made  in  which  were  used  the  cotyledonary  stalks  from  which  the 
plumules  had  been  cut  a  day  or  two  earlier.  Measurements  as  follows 
were  obtained  at  17°  to  19°  C. 

TABLE  115. 

p.  ct. 

Distilled  water 6 

Citric  acid,  0.01  N 4 

Sodium  hydroxid,  0.01  M 7 

Potassium  chloride,  hydrochloric  acid,  0.01  M 7 


Some  Hydration  Reactions  and  Growth.  153 

Another  trio  of  sections  from  an  older  stem  measuring  5  mm.  in 
diameter  was  taken  at  midday,  and  when  immersed  in  distilled  water 
at  21°  C.  increased  7.5  per  cent.  The  entire  lot  of  observations  con- 
firms and  supports  the  conclusion  that  the  stems  of  Helianthus  have 
their  hydration  capacity  more  nearly  satisfied  in  the  morning  than  at 
noon,  when  the  leaves  may  be  in  a  wilting  condition.  This  fact  would 
inevitably  have  an  important  influence  on  the  rate  at  which  such  stems 
might  elongate. 

The  growth  of  stems  of  Helianthus  was  measured  on  stems  growing 
in  the  soil  of  a  large  bed.  Heavy  wooden  bases  were  placed  on  the 
surface  of  the  soil  of  the  greenhouse  bench  and  the  stems  were  brought 
closely  against  this  and  fastened  at  the  base  of  the  growing  internodes 
in  such  manner  that  only  the  elongation  above  this  point  would  be 
registered  by  the  auxograph,  and  the  movements  of  the  base  due  to 
softness  of  soil  or  other  features  would  have  no  effect.  The  growing 
part  consisted  of  one  internode  approaching  maturity  and  a  terminal 
one  less  than  3  cm.  in  length.  A  fine  wire  loop  was  passed  around  this 
and  carried  up  over  the  arm  of  the  auxograph  lever. 

Temperatures  were  taken  by  thermometers  with  thin  bulbs  thrust 
into  the  stems  of  similar  plants  within  a  few  centimeters  of  the  one 
being  measured.  As  an  example  of  the  rate,  the  older  and  the  younger 
internodes,  together  having  a  length  of  about  15  cm.,  increased  2.7  mm. 
during  an  hour  at  midday  at  a  temperature  of  30°  C.,  while  in  the  2 
hours  immediately  afterwards,  when,  as  will  be  seen,  the  stem  of  another 
plant  was  showing  shrinkage  in  thickness,  the  rate  was  but  1.2  mm. 
per  hour  at  29°  C.  During  the  next  4  hours  the  temperature  slowly 
fell  to  19°  C.,  but  the  rate  of  elongation  came  up  to  2.1  mm.  per  hour, 
a  fact  plainly  due  to  decreased  water-loss. 

A  similar  behavior  ensued  on  the  following  day,  when  the  rate  was 
2.4  mm.  per  hour  at  midday  at  29°  to  30°  C.,  then  fell  off  to  1.4  mm. 
per  hour  during  the  next  two  hours  at  26°  C.,  and  then  to  0.6  mm.  per 
hour  during  the  following  2  hours.  Such  diminished  growth  might 
be  attributed  to  the  falling  temperature  if  it  had  not  been  observed 
that  a  higher  rate  was  shown  at  temperatures  as  low  as  14°  to  16°  C. 
This  lowered  rate  in  the  afternoon  was  accompanied  by  a  distinct 
wilting  of  the  leaves. 

An  auxograph  was  now  provided  with  a  cork  bearing  hollowed  to 
fit  against  a  stem  about  15  cm.  from  the  apex,  and  the  stem  was  held 
firmly  in  place,  so  that  any  variation  in  thickness  would  be  expressed 
by  the  free  arm  of  the  auxograph  and  traced  on  the  revolving  cylinder 
by  the  pen.  The  daily  action  may  be  exemplified  by  the  following 
transcript  from  the  notebook : 

"  Feb.  8.  The  temperature  had  risen  from  about  16°  C.  in  the  morning  to 
23°  C.  at  10  a.  m.,  at  which  time  an  increase  in  the  thickness  of  the  stem  at  a 
point  15  cm.  from  the  tip  had  been  in  progress  for  4  hours.  The  pen  was 


154 


Hydration  and  Growth. 


stationary  at  midday,  with  a  stem  temperature  of  26°  to  29°  C.  Actual 
shrinkage  now  began  and  continued  through  the  afternoon,  but  all  action 
ceased  at  night.  On  the  following  day  swelling  or  increase  in  thickness  began 
at  8  a.  m.  at  a  temperature  of  12.5  °C.,  but  continued  for  an  hour  only.  The 
leaves  were  beginning  to  flag  at  10h30m  a.  m.,  as  the  plants  had  not  been 
watered,  and  shrinkage  was  in  progress  before  noon  at  a  temperature  of 
23.5°  C."  (See  fig.  38.) 


FIG.  38. — Detail  of  arrangement  for  recording  variations  in  thickness  of  stem  of  Helianthus. 
A,  stem;  B  and  C,  parts  of  clamp  holding  stem  rigidly  in  place;  D,  support;  E,  cork  bearing 
of  short  lever  of  auxograph;  F,  pen  arm  of  auxograph,  and  G,  rack-and- pinion  column  of 
auxograph. 

A  second  preparation  was  set  up  on  February  10,  in  which  the 
variations  in  thickness  were  taken  at  a  place  but  6  cm.  from  the  tip  of 
the  stem.  The  adjustment  had  been  completed  by  night  and  an  in- 
crease in  thickness  of  0.38  mm.  took  place  in  12  hours  at  temperatures 
between  24°  and  18°  C.  Constant  readjustment  was  necessary  to 
obtain  reliable  measurements,  and  on  February  12  another  record  was 
obtained,  at  which  time  an  enlargement  of  0.4  mm.  was  recorded 
between  11  a.  m.  and  2h30m  p.  m.  at  temperatures  of  22°  to  25°  C. 
After  this  time  a  slight  shrinkage  occurred,  although  the  plant  was  so 
well  supplied  with  water  as  to  show  no  indications  of  wilting.  Here, 
as  in  the  leaves  of  Mesenibryanthemum,  elongations  may  be  taking 
place  at  a  lessened  rate  in  the  extreme  terminal  part  of  a  stem  in  which 
the  hydration  capacity  is  kept  continuously  higher  than  in  older  inter- 
nodes,  while  at  the  same  moment  an  actual  decrease  in  thickness  may 
be  taking  place  within  a  few  centimeters  of  the  elongating  active  zone. 
This  shrinkage  may  ensue  in  a  section  of  the  stem  which  has  not  lost 
the  capacity  for  elongation  altogether,  so  that  its  daily  record  shows  a 
period  of  elongation  at  a  moderate  rate  during  a  part  of  the  day,  then 
a  cessation  due  to  the  depletion  of  the  water-balance.1  It  is  obvious 
that  the  action  in  question  is  one  which  may  be  responsible  for  many 
mistaken  generalizations  bearing  upon  cessation  of  growth  and  effect 
of  temperature  on  growth  (fig.  39).  Attempts  at  interpretation  of  the 

*See  Brown  and  Trelease.     Alternate  shrinkage  and  elongation  of  growing  stems  of  Cestrum 
nocturnum.     Philipp.  Jour,  of  Science,  13:  No.  6,  333.     1918. 


Some  Hydration  Reactions  and  Growth. 


155 


rate  and  course  of  growth  of  any  plant  with  differentiated  tissues  which 
does  not  take  into  account  the  mechanical  composition  of  the  organs, 
and  especially  the  arrangement  of  the  growing  cell-masses  with  respect 
to  mature  parts,  may  encounter  many  pitfalls  and  can  hardly  fail  to 
be  inadequate. 


5 

15 
25 
35 
45 
55 
65 
75 
85 
95 


Feb.22 

8a.m. 


I5°C. 


X5 


I2p.m.     m.       12p.m.     m.       l?p.m.     m.       12p.m.    m.       12p.m.     m. 


FIG.  39. — The  upper  part  of  the  figure  shows  the  course  of  elongation  of  a  stem  of  Helianthus 
annuus  for  24  hours  beginning  at  5  p.  m.,  with  temperatures  of  the  plant  as  indicated,  X  5. 
Increase  in  length  denoted  by  downward  movement  of  the  pen.  Shown  on  a  scale  of  milli- 
meters as  indicated,  the  total  elongation  during  the  peiiod  being  19  mm.  The  lower  tracing 
shows  variations  in  thickness  of  a  stem  of  Helianthus  15  cm.  from  apex,  the  increase  being 
denoted  by  the  upward  movement  of  the  pen,  with  temperatures  of  the  plant  as  indicated. 
Shrinkage  or  cessation  of  enlargement  began  after  midday,  but  increase  was  again  mani- 
fested by  evening.  The  variation  is  amplified  30  times  and  is  shown  on  a  millimeter  scale, 
the  actual  increase  during  six  days  being  about  0.6  mm. 

Opportunity  for  the  measurement  of  growth  in  another  type  of 
structure  was  presented  by  the  legumes  of  Phaseolus  cultivated  in  the 
glass-house  of  the  Desert  Laboratory  in  April  1918.  These  pods  are 
first  measurable  when  they  have  attained  a  length  of  about  3  cm.  and 
a  thickness  of  2  mm.,  and  as  they  attain  a  final  length  of  10  to  12  cm. 
in  a  week,  the  rate  is  rapid  enough  to  afford  ready  means  of  detecting 
variations  and  connecting  them  with  possible  modifying  agencies. 
The  thickness  of  a  mature  pod  through  a  full-sized  bean  may  be  as 
much  as  6  to  8  mm.  The  imbibition  or  swelling  capacity  of  the  entire 
structure  and  its  contents  was  tested  at  two  different  stages.  The 
measurements  of  this  capacity  in  the  earlier  stage  was  made  upon 
sections  of  the  pod  less  than  a  centimeter  in  length,  which,  by  their 
bulging  contour,  showed  the  presence  of  an  embryo  bean  inside,  al- 
though this  was  much  less  than  a  millimeter  in  diameter  and  probably 
played  a  very  small  part  in  the  swelling.  The  increases  of  such  sec- 
tions at  two  different  temperatures  were  as  noted  in  table  116,  the 
average  thickness  of  the  trios  of  sections  being  2.7  to  2.8  mm. 


156 


Hydration  and  Growth. 


It  will  be  seen  by  reference  to  the  records  of  growth  cited  in  table  116 
that  the  higher  temperature  lies  above  the  point  at  which  the  most 
rapid  elongation  or  thickening  takes  place,  a  matter  which  might  be 
due  to  excessive  water-loss  or  to  the  action  of  residual  acids  at  high 
temperatures. 

TABLE  116.  TABLE  117. 


Swelling  of  bean 
pods. 

18°  C. 

38°  C. 

p.  ct. 

p.  ct. 

Distilled  water  

2 

2.7 

Citric  acid,  0.01  N.  . 

2 

Shrinkage. 

Swelling  of  beans. 

18°  C. 

38°  C. 

p.  ct. 

p.  ct. 

Distilled  water  

11 

10.6 

Citric  acid,  0.01  N.  . 

10.4 

5.5 

Beans  nearly  mature  but  still  in  the  process  of  enlargement  were 
removed  from  green  pods,  the  ends  of  the  cotyledons  cut  away,  and 
then  a  slice  removing  the  hypocotyl;  the  remainder  of  the  cotyledons 
came  away  free  from  the  outer  coating  or  mem- 
brane.   The  average  diameter  of  trios  of  such  sec- 
tions was  3  to  3.2  mm.,  and  their  swelling  was  as 
given  in  table  117. 

The  amount  of  hydration  was  less  at  the  higher 
temperature  in  distilled  water,  suggesting  that  the 
point  of  maximum  imbibition  or  swelling  lies  be- 
low 38°  C.  In  the  presence  of  acid  the  amount  of 
water  absorbed  is  distinctly  less  than  at  18°  C. 
These  data  being  available,  attention  may  now  be 
profitably  turned  to  the  features  of  enlargement  of 
the  pods. 

Preparations  were  made  by  which  delicately 
weighted  auxographs  recorded  the  variations  in 
thickness  of  pods  in  the  stage  when  about  half  the 
final  length  had  been  reached.  The  end  of  the 
bearing-lever  rested  over  an  enlarging  bean,  and 
variation  during  a  week  is  shown  in  figure  40. 

The  localization  of  growth  had  been  previously 
determined  by  the  well-known  expedient  of  mark- 
ing a  young  pod  which  was  in  the  stage  of  initial 
growth  of  the  young  beans  into  four  1-centimeter 
intervals  (fig.  41).  Swelling  tests  had  been  made 
of  the  pods  in  this  stage  (table  116).  Ten  days 
later  the  basal  and  apical  intervals  had  increased 
to  2.5  cm.,  while  the  other  two  had  each  elongated 
to  4  cm.  All  measurements  of  variations  in  thick- 
ness were  made  in  this  median  region  of  maximum  elongation  (fig.  42). 

Growth  both  in  length  and  thickness  of  the  young  pods  was  at  the 
lowest  rate  at  night,  during  which  period  the  temperature  was  at  15°  C. 


FIG.  40. — Arrangement  of 
vertical  lever  of  auxo- 
graph  making  record 
of  variations  in  thick- 
ness of  growing  bean 
and  pod. 


Some  Hydration  Reactions  and  Growth. 


157 


or  below.  As  the  temperature  of  the  air  rises  above  this  point  in  the 
morning,  acceleration  ensues  and  a  high  rate  prevails  until  the  ther- 
mometer shows  25°  C.  or  above,  at  which  time  retardation  takes  place, 
which  may  continue  to  complete 
cessation  or  even  shrinkage.  It  was 
noted  that  the  behavior  with  respect 
to  the  temperature  in  this  region 
might  be  modified  by  varying  the 
conditions  of  water-loss,  and  that 
growth  might  be  maintained  at  a 
higher  rate  if  high  humidity  around 
the  growing  member  is  maintained. 
As  the  temperature  falls  at  the 


FIG.  41. — Diagram  showing  the  elonga- 
tion of  four  1-cm.  intervals  into  which 
a  young  pod  was  divided.  Maximum 
increase  ensued  in  third  centimeter 
from  base. 


FIG.  42. — Arrangement  of  auxograph  to 
take  variations  in  length  of  pod  of 
Phaseolus.  The  mature  tip  of  pod  is 
fastened  to  cork  buffer  on  end  of  lever  of 
instrument. , 


close  of  the  day  the  rate  accelerates  again  and  growth  is  rapid  until 
checked  by  the  falling  temperature.     (Fig.  43.) 

These  records  were  all  made  of  plants  not  subjected  to  the  direct' 
action  of  the  sun.  One  was  placed  in  such  position  that  the  sunlight 
fell  directly  upon  the  pod  for  about  15  minutes  before  6  p.  m.,  with  the 


m.     tfpnti.    m.     12p.m.    m.    12p.m.     m.     12p.m.    m.     12p.m.   m.     I2.p.m.    m.     12p.m.    m. 


FIG.  43. — Tracing  of  auxographic  record  of  growth  in  length  of  bean  pod,  X  45.  The  features 
of  active  elongation  are  similar  to  those  described  for  variations  in  thickness  in  fig.  44. 
Retardation  of  growth  is  seen  at  temperatures  above  30°  C.  Downward  course  of  pen 
denotes  increase. 

result  that  a  sudden  enlargement  followed,  which  was  quickly  retracted 
and  quiescence  or  slow  shrinkage  followed.  Such  sudden  variations 
have  been  seen  in  other  types  of  organs,  such  as  the  joints  of  Opuntia, 
and  seem  to  be  enlargements  due  to  expansion  of  gases  in  the  organ, 
the  cavity  of  the  pods  in  this  case  being  of  such  size  that  a  marked 
response  might  be  expected  (fig.  44).  The  features  of  variation  of 


158 


Hydration  and  Growth. 


thickness  are  recognizable  in  changes  in  length,  although  the  action 
of  120  mm.  of  tissue  is  involved  as  against  2  or  3  mm.  in  the  measure- 
ments of  thickness.  The  structural  arrangement  of  the  cell-masses 
and  the  shape  of  the  cavity  of  the  pod  would  operate  to  minimize  the 
shrinkages  so  apparent  when  thickness  is  measured. 

i2p.m.      m.        12p.m.       m.         12p.m.      m. 


15 

/ 

/ 

/         ; 

/          •'      ' 

/ 

">c. 

/      I6°C 
1~  \ 

/     7 

/        ; 
( 

| 

•3K 

1 

^     f~i?£sJ 

t,  r 

X45 

i 

28°C.            \ 

i 

I 

FIG.  44. — Tracing  of  an  auxographic  record  of  growth  in  thickness  of  pod  of  Phaseolus.  Down- 
ward movement  of  the  pen  denotes  increase  in  thickness,  X  45.  Temperatures  given  are 
of  the  air  near  plants.  The  sudden  shrinkage  between  5  and  6  p.  m.  took  place  during 
a  brief  daily  illumination  by  direct  rays  of  sun.  Scale  ruled  to  5  mm.  and  12-hour  inter- 
vals. Summer-time  schedule.  (See  fig.  40  for  illustration  of  the  arrangement  of  auxo- 
graphic levers.) 

A  pair  of  tests  was  now  arranged  in  which  one  pod  was  placed  in- 
side a  cell  consisting  of  a  short  section  of  a  glass  T-tube  of  about  1  cm. 
internal  diameter.  The  pod  was  placed  in  a  horizontal  position  in 
the  main  section  of  this  tube,  which  rested  solidly  on  a  concrete 


f     WHMW  \VVW\V 


FIG.  45. — Glass  chamber  for  controlling  humidity  in  making  auxographic  record  of  pod  of  Pha- 
seoliis.  Ends  of  horizontal  part  of  chamber  closed  by  cork  stoppers  fitted  to  stem  of  pod 
and  thermometer.  Tube  to  be  closed  around  vertical  arm  of  instrument  with  cotton  wool. 

block.  The  end  around  its  stem  was  closed  loosely,  and  the  opposite 
end  of  the  tube  held  a  cork  and  small  thermometer.  The  vertical 
arm  of  the  auxograph  reached  its  bearing  on  the  pod  through  the 
upright  arm  of  the  T-tube  and  opportunity  was  given  to  keep  record 


Some  Hydration  Reactions  and  Growth. 


159 


of  the  temperature  of  the  air,  which  in  this  setting  must  have  been 
practically  identical  with  that  of  the  inclosed  pod  (fig.  45).  The  air 
inside  this  chamber  was  at  a  high  degree  of  humidity,  and  it  was 
found  that  the  afternoon  cessation  or  retardation  of  growth  was  not 
so  marked  in  actively  growing  pods  and  that  it  did  not  come  on  so 
early  in  the  development  of  the  pod  as  in  those  exposed  to  the 
evaporating  influence  of  the  freely  circulating  air.  The  influence  of 
high  humidity  approaching  saturation  was  shown  more  directly  by  the 
application  of  wet  slips  of  filter-paper  in  such  manner  that  the  pod 
was  completely  swathed  and  evaporation  reduced  to  a  minimum. 
This  treatment  was  also  successfully  applied  to  pods  resting  upon  a 
cork  base  and  not  inclosed  in  the  chamber.  When  a  slowly  growing 
young  pod  was  thus  given  an  atmosphere  of  high  humidity  at  tem- 
peratures from  20°  to  27°  C.  no  alteration  in  the  rate  would  be  visible 
for  nearly  an  hour,  but  at  the  end  of  this  time  an  abrupt  acceleration 
would  ensue  which  would  continue  for  as  much  as  2  hours,  and  then, 
if  the  supply  of  moisture  were  not  renewed,  a  slackening  would  ensue 
which  would  bring  the  rate  back  to  the  point  at  which  it  was  growing 
previous  to  the  treatment  (see  figs.  46  and  47). 


•n.      12  p.m.      i 

n.       12  p.m. 

7i.       12  p.m.     r 

n.       12p.m. 

n.       12p.m. 

TI.       12p.m.    .  m. 

i 

1 

1 

^5 

»-  1 
I 

^\   ,' 

1 
j 

i 

i 
I 

l"  

1 

.     i 

i 

1 
1 

i 
i 

H^ 

__^    jir'C. 

30°C.l      |nor 

^ 

\            \  X45 

\ 

\ 

r30°ai~  —  - 

—  •*•-,     "-*  •-" 
Y      27^^ 

f  3°"C<          1 

\            \ 

\            \ 

\            \ 

\            V 

\             \ 

\           '\            \ 

FIG.  46. — Tracing  of  auxographic  record  of  pod  of  Phaaeolua  which  was  2  mm.  in  thickness  at 
beginning  of  record.  Downward  movement  of  pen  denotes  increase  in  thickness,  X  45. 
The  range  of  active  growth  lies  between  15°  and  30°  C.  and  consequently  acceleration  ensued 
in  the  morning,  retardation  occurred  as  temperatures  about  30°  C.  were  reached  in  the  after- 
noon, and  the  rate  increased  again  at  sunset,  when  the  temperature  fell  to  a  point  below  30° 
C.,  but  slowing  down  followed  in  the  cooler  night  temperatures. 

12p.m.       m.          I2p.m.      m.          12p.m.      m.         12p.m.      m. 


5 
15 

//'/,'              /'//// 

/     '     T  —  ^^—  /—   (/    21     !'  3-l^ 

1            I            1            1            1            111! 

FIG.  47. — Tracing  of  auxographic  record  of  variations  in  thickness  of  young  pod  of  bean.     The 
enlargements  caused  by  humidity  are  seen  at  1,  2,  and  3. 

The  greater  part  of  the  enlargement  registered  in  the  above  measure- 
ments was  due  to  the  growth  of  the  beans,  and  the  imbibition  capacity 
of  such  seeds  has  been  measured  separately.  A.  Dachnowski  (see 
reference,  p.  63)  found  that  mature  seeds  absorbed  and  held  more 
water  in  acids  and  hydroxids  than  in  distilled  water  at  temperatures 
not  given,  and  that  the  amounts  taken  up  in  alkaline  solutions  was 
greater  than  that  in  acids.  The  general  imbibition  reactions,  in  my 
own  experiments,  of  young  seeds  which  had  reached  a  thickness  of 


160  Hydration  and  Growth. 

2.8  mm.  to  3  mm.,  is  shown  by  the  measurements  of  swellings,  made 
at  15°  to  16°  C.,  shown  in  table  118. 

TABLE  118. 

p.  ct. 

Distilled  water 8.2 

Citric  acid,  0.01  N 5.4 

Potassium  hydroxid,  0.01  M 12.5 

Potassium  nitrate,  0.01  M ;  citric  acid,  0.01  N 8.2 

Actually  lessened  imbibition  took  place  in  acid  as  compared  with  that 
in  water;  the  addition  of  equimolecular  solution  of  potassium  nitrate 
to  acid  brought  the  swelling  up  to  that  hi  water.  The  total  absorbed 
in  alkali  was  markedly  greater  than  in  any  solution  tested. 

The  measurements  of  variations  in  length  and  thickness  of  the  suc- 
culent leaves  of  Mesembryanthemum,  stems  of  Helianthus,  and  of  the 
pods  of  Phaseolus,  and  the  flattened  stems  of  Opuntia,  yield  ample 
evidence  that  the  fluctuations  hi  growth  show  a  direct  relation  to  the 
hydration  capacity  of  the  growing  cell-masses,  and  that  as  a  morpho- 
logically complex  member  or  organ  approaches  maturity,  the  fully 
developed  tissues  show  a  varying  water  capacity  different  in  many 
respects  from  that  of  the  embryonic  cell-masses.  Some  of  the  irreg- 
ularities hi  the  course  of  growth  of  internodes  are  due  to  the  fact 
that  these  members  include  regions  of  embryonic  tissue  and  tracts  hi 
all  stages  of  differentiation  approaching  maturity. 


XII.  WATER-CONTENT,  DRY  WEIGHT,  AND  OTHER 
GENERAL  CONSIDERATIONS.- 

Two  different  types  of  organs  or  shoots  with  respect  to  the  variations 
in  the  water-content  and  dry  weight  are  recognizable  in  the  material 
which  has  served  for  studies  in  growth  as  described  in  this  volume  and 
in  the  work  of  other  writers.  The  commoner  types  of  woody  stems, 
of  thin  leaves,  and  of  the  organs  of  the  greater  number  of  the  higher 
plants  undergo  a  development  which  terminates  in  a  mature  stage  in 
which  the  proportion  of  solid  material  is  very  much  higher  than  that 
found  in  younger  material.  A  parallel  procedure  is  the  prevalent  one 
in  the  tissues  of  the  higher  animals.  Thus,  by  way  of  illustration, 
Donaldson  found  that  the  proportion  of  water  in  the  bodies  of  mammals 
diminishes  with  age,  and  Hatai  has  shown  that  the  percentage  of  water 
is  an  indicator  of  phases  of  chemical  alteration  in  the  composition  of 
the  body.1 

Growth  and  differentiation  of  cell-masses  into  specialized  tissues  is 
not  inseparably  connected  with  increases  in  dry  weight,  however,  as 
has  been  demonstrated  by  studies  of  the  growth  of  frog  larvae2  in  the 
earlier  stages,  and  it  is  highly  probable  that  similar  phenomena  are 
prevalent  in  the  fleshy  fungi  and  other  lower  forms  of  plants. 

The  distinction  between  the  two  kinds  of  growth  has  not  been  made 
previously  in  studies  of  plants,  and  the  matter  was  finally  taken  into 
consideration  in  the  experiments  late  in  1918.  Stems  of  Helianthus 
and  pods  of  Phaseolus  illustrate  the  kind  of  material  in  which  dry 
weight  increases  with  age,  upon  which  the  greater  part  of  all  studies 
in  growth  have  been  carried  out. 

Etiolated  plants  furnish  examples  of  growth  with  a  diminished 
increase  in  dry  weight.  Chief  interest  attaches  to  plants  which  nor- 
mally show  such  action,  and  the  most  striking  illustrations  are 
furnished  by  the  organs  of  succulent  plants  and  by  fruits.  The 
relative  amount  of  solid  material  in  the  flattened  joints  of  Opuntia  does 
not  increase  with  the  course  of  development  toward  maturity,  and 
joints  which  have  reached  full  size  may  contain  over  91  per  cent  of 
water.  Secondary  thickening,  especially  that  which  results  from 
branching  and  the  development  of  additional  fibrovascular  tissue, 
may  cause  an  added  amount  to  be  formed.  The  proportion  of  dried 
material  and  water  hi  the  leaves  of  Mesembryanthemum  does  not  vary 
greatly  with  age.  These  and  probably  all  succulent  forms  are  char- 
acterized by  an  exaggerated  production  of  mucilages  or  pentosans, 
and  have  certain  implied  cycles  of  metabolism,  including  an  incomplete 

1  Donaldson,  H.  The  relation  of  myelin  to  the  loss  of  water  in  the  mammalian  nervous  system 
with  advancing  age.  Proc.  Nat.  Acad.  Sc.,  2:  350.  1916.  Hatai,  S.  Changes  in  the  composi- 
tion of  the  entire  body  of  the  albino  rat  during  the  life  span.  Amer.  Jour.  Anat.,  1 :  23.  1917. 

1  Ostwald,  W.     Ueber  zeitlichen  Eigenschaften  der  Entwickelungsvorgange,  p.  49.     1908. 

161 


162  Hydration.  and  Growth. 

type  of  respiration  which  leaves  large  acid  residues.  These,  con- 
stituting the  total  acidity  of  the  cell-masses,  may  vary  greatly  during 
development  and  during  the  course  of  a  day,  and  the  actual  acidity  or 
hydrogen-ion  concentration  of  the  sap  resulting  from  the  buffer  situa- 
tion may  also  show  a  marked  variation,  but  within  narrower  limits. 

Although  the  development  and  maturation  of  fruits  such  as  berries 
obviously  includes  a  growth  in  which  the  total  effect  is  one  of  practical 
maintenance  or  increase  hi  the  water-content,  studies  of  their  growth 
seem  to  be  lacking.  It  was  therefore  planned  to  arrange  a  final  series 
of  experiments  in  which  the  enlargement  of  fruits  with  increasing  dry 
weights  and  with  small  and  more  nearly  constant  dry  weights  should 
be  measured.  The  walnut  was  taken  to  represent  a  structure  with 
accumulating  solid  matter  and  the  tomato  for  the  other  type. 

The  walnut  consists  of  a  thick,  fleshy  exocarp  and  a  heavy  endocarp 
which  finally  becomes  hard  and  bony  with  the  deposition  of  anhydrous 
wall  material.  The  inclosed  embryo  also  accumulates  a  large  amount 
of  condensed  food-material.  The  tomato  is  a  large  globose  berry  hi 
which  deposition  and  thickening  is  confined  to  the  small,  hard  seeds. 
The  greater  part  of  the  fruit  is  a  fleshy,  watery  pulp,  which  becomes 
more  highly  hydrated  as  progress  is  made  toward  maturity. 

Nuts  of  Juglans  californica  var.  querdna  Babcock,  of  various  sizes 
from  3  mm.  in  diameter  to  that  approaching  maturity,  were  borne  on  two 
trees  in  the  garden  at  Carmel,  California,  in  June  1918.  Suitable  supports 
being  provided,  the  bearing  lever  of  an  auxograph  was  rested  as  lightly  on 
the  young  nuts  as  was  consistent  with  a  clear  record,  and  temperatures 
were  taken  by  thin  thermometers  thrust  into  similar  nuts  or  into  young 
stems  near  the  preparation.  15  nuts  were  measured  for  periods  of  2 
or  3  days,  or  for  as  long  as  2  months  in  the  case  of  No.  10. 

Coincidently  with  the  measurements,  an  effort  was  made  to  determine 
the  degree  of  saturation  or  hydration  of  the  stems  on  which  the  nuts 
were  borne.  A  well-defined  "negative"  pressure  was  detected  hi  the 
basal  branches  of  Juglans  major,  which  was  growing  near  the  experi- 
mental tree.  A  basal  branch  1.2  meters  from  the  trunk  gave  a  dry- 
looking  surface  when  it  was  cut  off. 

A  section  of  a  similar  branch  about  8  mm.  in  thickness  and  42  cm. 
long  was  cut  away  from  another  basal  branch  of  the  tree,  the  end  of  the 
detached  portion  quickly  sealed  with  vaseline,  and  when  all  was  in  readi- 
ness the  tip  was  excised  and  the  cut  thrust  into  water  to  ascertain  the 
actual  deficiency  hi  this  portion;  14  hours  later  a  total  of  6  c.  c.  of 
water  had  been  absorbed  and  24  hours  later  8.5  c.  c.,  which  was  a  practi- 
cal saturation,  at  a  temperature  of  18°  to  20°  C.  The  volume  of  the 
branch  proved  to  be  35  c.  c.,  so  that  the  amount  of  water  absorbed  was 
24  per  cent  of  the  total. 

Sections  of  young  internodes  of  Juglans  californica  querdna  which 
had  an  average  diameter  of  about  2.5  mm.  were  swelled  in  solutions  as 


Imbibition  and  Growth  in  Fruits. 


163 


below,  then  dried,  and  swelled  again,  with  results  as  shown  hi  table  119 
at  16°  C.: 

TABLE  119. 


Swelling  of  sections  of  stems  of 
Juglans. 

Fresh 
living. 

After 
drying. 

Distilled  water  

p.  ct. 
10 

p.  ct. 
34 

Citric  acid,  0.01  N  

14 

34 

Potassium  hydroxid,  0.01  M  

13.2 

34 

Potassium  nitrate,  0.01  M  

12 

32 

(On  basis  of  original  thickness.) 

The  unsatisfied  water  capacity  of  these  sections  taken  from  young 
terminal  internodes  was  comparatively  great,  doubtless  due  hi  part  to 
the  constant  drain  of  the  active  leaves  they  bore.  The  older  wood,  in- 
cluding that  formed  the  previous  year,  showed  an  absorptive  capacity 
of  22  per  cent  hi  water.  It  is  from  these  older  internodes  that  the  nuts 
arise. 

The  nuts  were  highly  turgid,  exuded  sap  when  cut  into,  and  hence 
must  have  had  a  colloidal  composition  which  acted  to  withdraw  water 
from  the  stems,  which  were  less  highly  hydrated.  The  soil  was  low  in 
moisture-content  at  this  tune,  as  it  had  been  4  or  5  months  without 
rain. 

Tests  of  nuts  8  to  10mm.,  from  which  tangential  slices  had  been  removed 
to  give  a  uniform  thickness  of  7.5  mm.,  were  made  in  July,  and  these 
swelled  at  temperatures  of  17°  to  20°  C.  in  solutions  as  follows: 


TABLE  120. 


Distilled  water 

Citric  acid,  0.01  M 

Potassium  hydroxid,  0.01  M . 
Potassium  nitrate,  0.01  M .  . , 


p.  ct. 
1.4 
1.8 
1.4 
2 


A  useful  conception  of  the  hydration  conditions  in  the  stems  and 
fruits  may  be  formed,  if  due  weight  is  given  to  the  measurements  cited 
above.  The  woody  branches  of  the  previous  year,  on  which  both  the 
leafy  green  twigs  and  those  bearing  the  nuts  are  borne,  had  a  relatively 
large  deficiency  hi  water,  so  that  sections  a  few  centimeters  long  absorbed 
about  20  to  25  per  cent  of  their  volume  of  distilled  water  hi  24  hours  at 
20°  C.  No  swelling  test  was  made,  but  it  is  obvious  that  an  enlarge- 
ment of  only  a  small  fraction  might  be  shown  by  this  or  any  branch 
with  a  mature  woody  cylinder.  The  active  green  twigs  still  hi  a  state 
of  elongation  arising  from  these  branches  had  a  swelling  capacity  of  10 
per  cent.  The  growing  nuts  arising  from  the  drier  stems  exuded  water 
from  cut  surfaces,  the  cotyledons  being  sacs  of  watery  fluid,  hi  contrast 
to  the  dry  appearance  of  sections  of  the  youngest  internodes,  and  showed 
a  swelling  of  less  than  2  per  cent  and  soon  shrunk  when  placed  in  a  cylin- 


164  Hydration  and  Growth. 

der  of  distilled  water  after  being  cut  in  halves.  In  a  system  of  this  kind 
any  alteration  of  the  conditions  which  would  facilitate  transpiration 
would  have  a  differential  effect  on  the  older  stems,  the  green  leafy  twigs, 
and  the  fruits.  The  loss  from  the  stems  would  be  affected  least,  since 
the  bark  would  effectually  prevent  any  notable  increase  hi  evaporation 
from  the  relatively  dry  woody  tissues.  The  loss  from  the  leafy  twigs 
would  of  course  tend  to  become  greater  and  the  deficit  in  both  leaves 
and  twigs  would  be  increased  and  their  absorbing  power  correspondingly 
increased.  The  outer  integument  of  the  nuts  being  still  hi  an  embryonic 
condition  and  being  highly  hydrated,  the  loss  would  reach  a  maximum 
rate,  with  the  daily  effect  of  causing  a  cancellation  of  enlargement  begin- 
ning mid-forenoon  at  20°  to  22°  C.  and  continuing  until  mid-afternoon, 
when  a  fall  in  temperature  brought  transpiration  to  a  rate  below  that  of 
accession  from  the  stem. 

A  large  percentage  of  the  nuts  which  were  placed  under  the  auxo- 
graph  lever  were  cast  off  at  various  stages  of  development  by  abscission 
of  the  stalk.  The  inciting  causes  of  the  anatomical  change  which 
results  in  abscission  lie  outside  the  scope  of  this  article.  It  was 
noted,  however,  that  it  was  preceded  by  a  period  in  which  the  nut 
showed  a  shrinkage  by  day  in  the  higher  temperatures  and  lessened 
humidity,  alternating  with  equalizing  enlargements,  at  nights.  Fin- 
ally, an  abrupt,  rapid,  and  continuous  shrinkage  resulted  in  the  separ- 
ation of  the  stalk. 

The  general  features  of  growth  of  these  nuts  may  be  illustrated  by  a 
re'sume'  of  history  of  No.  10,  which  was  under  continuous  observation 
from  July  15  to  September  9,  1918,  during  which  period  of  56  days  its 
diameter  increased  from  16  mm.  to  26.5  mm.  Of  this,  2.25  mm.  was 
gained  in  the  first  5  days  of  cool,  foggy  weather.  This  effect  was  con- 
firmed by  the  fact  that  a  cessation  or  retardation  occurred  at  midday 
and  was  most  pronounced  on  hot,  sunny  days,  suggesting  a  direct 
water-loss.  In  the  week  ending  July  29  the  total  growth  was  an  in- 
crease of  1.7  mm.  This  period  was  characterized  by  heavy  fogs  and 
mists  in  the  forenoon,  both  the  amount  of  shrinkage  and  rate  of  in- 
crease being  lessened — an  equalization  to  be  ascribed  in  part  to  ap- 
proaching maturity.  The  temperature  taken  from  a  thermometer 
thrust  in  a  young  branch  of  the  thickness  of  the  nut  ranged  from  13° 
to  22°  C.  The  completion  of  the  record  of  No.  10  was  followed  by  cut- 
ting of  the  branch  bearing  it  at  a  distance  of  30  cm.,  placing  the  excised 
end  in  water,  and  arranging  the  entire  preparation  in  the  dark  room  at 
17°  C.,  with  the  nut  under  the  bearing  lever  of  the  auxograph.  Swell- 
ing continued  for  about  20  hours,  after  which  shrinkage  began,  which 
rapidly  accelerated  (see  fig.  48). 

The  general  features  of  growth  are  also  well  illustrated  by  the 
following  notes  on  No.  15,  which  was  brought  under  observation  when 
it  was  about  15  mm.  in  diameter  and  put  under  an  auxograph  ampli- 


Imbibition  and  Growth  in  Fruits. 


165 


fying  45  on  August  3.  Great  daily  variations  in  size,  with  a  net  total 
increase,  were  displayed  every  day.  Usually  enlargement  could  be 
detected  between  noon  and  2  o'clock,  which  continued  until  8  or  10 
the  following  morning.  If  the  sun  rese  clear,  shrinkage  began  imme- 
diately. If  the  morning  was  foggy  it  would  be  delayed.  Minor  vari- 
ations might  be  brought  about  by  the  shade  of  clouds,  especially  notice- 
able at  noonday  August  6  and  to  be  seen  at  other  times. 


12p.m. 


I2o.m.      m.         12  p.m.      m. 


I5-22°C.  during  this  beriod 


25  I- 

35 


12p.m. 


5 

15 
25 

/ 

SEPT.  9,  1918       Or/e  day  in  dark  room  at  I7°C. 
/                X  20        Swelling  for  20  hours 

Branch  30  cm 
loni  in  vessel 

-.  /                                 then  slight  shrinkage 

of/water 

-t—  ^—                         —  ;           — 

FIG.  48. — Variations  in  volume  of  nut  of  Juglans  during  56  days.  Enlargement  is  denoted  by 
downward  course  of  pen  tracing,  X  10.  The  lowermost  section  of  the  figure  gives  auxographic 
record  of  swelling  of  a  nut  in  a  dark  room  on  a  branch  30  cm.  in  length  with  the  cut  end  in  a 
vessel  of  water.  Swelling  for  20  hours  occurred  after  shrinkage  began  as  denoted  by  upward 
course  of  pen  tracing. 

After  these  facts  were  noted,  experimental  modifications  were 
arranged.  Temperatures  were  taken  from  a  branch  16  mm.  in  thick- 
ness which  were  probably  within  a  degree  of  that  of  the  nut  at  all 
times.  A  screen  was  arranged  to  cut  off  the  direct  rays  of  the  sun  at 
midday,  the  nut  being  exposed  for  about  4  hours  in  the  forenoon  to  di- 
rect illumination.  The  temperatures  ranged  from  14°  to  25°  C.  The 
occurrence  of  fogs  and  of  rain  added  to  the  variations  in  the  conditions 
affecting  transpiration.  The  shrinkage  in  the  forenoon  was  abrupt 
and  marked,  being  lessened  on  foggy  days,  and  reaching  an  extreme  of 
4  mm.  when  the  temperature  rose  from  14°  to  25°  C.  in  the  4  hours, 
while  it  was  on  no  day  less  than  one-fourth  this  amount.  The  increase 
varied  from  a  minimum  growth  of  less  than  0.1  mm.  on  a  cool,  foggy 


166 


Hydration  and  Growth. 


day  to  0.7  mm.  when  shaded  on  August  6,  and  to  a  similar  amount  in 
a  rain  on  September  11,  at  which  time  it  was  in  an  advanced  state  of 
development  (fig.  49). 

It  is  to  be  seen  from  the  above  that  the  fruit  of  the  walnut  in  an 
environment  favorable  to  its  development  exhibits  daily  variations  in 
growth  clearly  attributable  to  the  balance  between  transpiration  and 
absorption.  The  nut  in  a  growing  condition  has  a  high  water-content 
and  a  small  unsatisfied  capacity,  but  its  supply  from  the  relatively 
dry  stems  must  come  slowly — so  slowly  that  any  marked  increase  in 
transpiration  would  overbalance  the  absorption  by  the  nut  and  result 
in  cessation  of  enlargement  or  even  shrinkage. 


IZp.m.      m.       Ifp.m.       m          12p.m.      m.        12p.m.       m.       12p.m.      m.      12p.m. 


/         NtylS         i      Jug|ans  Calif. quqlrcina   / 


FIG.  49. — -Variations  in  volume  of  a  growing  nut  of  Juglana  15  mm.  in  diameter  at  beginning 
for  a  period  of  45  days.  The  marked  acceleration  under  the  conditions  of  high  humidity 
and  abundant  water-supply  are  illustrated  in  the  record  beginning  September  9.  Retarding 
or  shrinking  effects  of  noonday  temperature  and  low  humidity  and  masking  effects  of  fog 
are  also  illustrated. 

The  fruit  of  the  tomato  (Lycopersicori)  presents  features  of  water- 
content  unlike  any  other  organ  the  growth  of  which  had  been  under 
observation  in  present  studies.  The  most  striking  feature  of  this 
phase  of  the  matter  is  that  the  proportion  of  solid  material  is  higher  in 
young  fruits  than  in  mature  ones.  In  the  determination  of  the  pro- 
portions, first  young  fruits  less  than  a  week  old  were  taken  and  4 
tomatoes  with  radial  diameters  of  14,  16,  17,  and  18  mm.  were  found  to 
weigh  14.650  grams.  These  were  fragmented  and  placed  in  a  beaker 
on  a  water-bath  at  about  100°  C.  for  48  hours,  at  which  time  the  dry 
material  remaining  was  1.90  grams.  From  this  it  is  to  be  seen  that  the 
young  fruit  contained  87  per  cent  of  water  and  13  per  cent  of  dry 
material.  A  mature  fruit  of  the  same  kind  as  those  measured  was 
46  mm.  in  axial  diameter  and  58  mm.  in  radial  diameter  and  weighed 
93.050  grams.  This  was  dried  over  water-bath  for  2  days,  at  which  time 


Imbibition  and  Growth  in  Fruits.  167 

8.400  grams  remained.  From  this  it  is  to  be  seen  that  the  ripe  fruit  con- 
tained 91  per  cent  of  water  and  9  per  cent  of  dry  material.  In  fact, 
these  fruits  show  a  better  parallel  to  the  hydration  reactions  of  the 
prepared  biocolloids  than  any  living  material  which  has  hitherto  been 
examined  for  the  purpose  of  estimating  the  value  of  the  physical  fac- 
tors in  growth. 

A  number  of  plants  of  the  tomato  were  grown  in  suitable  boxes  of 
soil  at  a  ranch  in  the  Carmel  Valley,  and  were  in  such  a  stage  of  de- 
velopment that  young  fruits  were  available  at  the  Coastal  Laboratory 
early  in  August  1918.  Six  plants  in  all  were  used  and  continuous  rec- 
ords from  fruits  of  an  axial  diameter  of  3  to  4  mm.  to  maturity  at  50  to  55 
mm.  were  obtained.  The  fruits  were  obla,te-spheroid  in  form  and  the 
auxograph  was  arranged  to  register  increase  in  diameter  nearly  par- 
allel to  the  axis  in  some  cases  and  radially  or  at  right  angles  to  it  in 
others.  In  addition  to  the  other  advantageous  features  of  this  mate- 
rial, the  regular  form  and  mode  of  growth  made  it  possible  to  use  the 
Variations  in  diameter  as  a  basis  for  calculating  the  changes  in  volume 
of  the  fruits  taken  as  spheres. 

Temperatures  were  taken  by  thrusting  the  thin  bulbs  of  small 
thermometers  into  fruits  near  the  one  tinder  measurement.  The 
development  of  such  fruits  was  but  little  affected  by  this  wounding 
and  the  thermometers  remained  firmly  in  place,  as  in  the  fleshy  joints 
of  Opuntia,  in  the  measurement  of  which  this  method  was  first  practiced. 
The  preparations  stood  in  a  well-ventilated  glass-house  and  the  soil 
around  the  roots  was  kept  moist  in  accordance  with  the  cultural  re- 
quirements of  these  plants.  The  results  may  be  best  set  forth  by  the 
description  of  the  action  of  the  several  fruits  measured. 

No.  1  was  placed  in  the  greenhouse  and  a  fruit  29  mm.  in  diameter 
was  fixed  on  a  block  of  hard  cork  in  such  position  that  it  gave  a  radial 
bearing  to  the  auxograph,  which  was  set  to  amplify  changes  in  volume 
by  5,  on  August  9.  The  record  was  kept  continuously  until  September 
18,  at  which  time  the  radial  diameter  of  the  fruit  was  51.5  mm.  The 
fruit  was  turning  yellow  on  September  16  and  was  showing  fluctuations 
in  volume  comparable  to  those  in  No.  2,  with  which  it  was  run  in  close 
comparison  and  under  almost  exactly  the  same  conditions  of  moisture 
and  temperature  as  recorded. 

No.  2  was  adjusted  to  the  auxograph  in  the  greenhouse  on  August  9, 
in  such  manner  as  to  give  modifications  of  the  axial  diameter,  which 
at  this  tune  was  about  27  mm.  The  record  was  continuous  until 
September  18,  at  which  time  the  diameter  was  50.5  mm.  This  fruit, 
like  No.  1,  was  beginning  to  turn  yellow  on  September  16. 

No.  3,  10  mm.  in  diameter,  was  adjusted  to  the  auxograph  to  record 
variations  in  radial  diameter  on  August  21,  and  a  record  was  kept 
continuously  with  frequent  notations  of  temperature  and  sunshine, 
etc.  It  is  to  be  noted  that  1,  2,  and  3  were  under  equable  temperatures, 


168 


Hydration  and  Growth. 


19°  to  20°  C.,  and  high  relative  humidity  during  the  rainfall  of  Sep- 
tember 11  and  12. 

The  fact  that  the  greatest  increase  in  growth  occurs  in  fruits  at 
diameters  between  16  and  25  mm.  hi  diameter,  before  half  the  final 
size  is  reached,  is  a  point  to  which  we  shall  recur  in  the  discussion  of 
growth  in  terms  of  volume.  Thus,  in  No.  3  the  increases  in  thickness 
weekly  were  as  follows:  6  mm.,  6.3  mm.,  2.5  mm.,  3.5  mm.  (fig.  50). 


I2p.m.    m.      12p.m.      m.      12p.m.     m.       12p.m.     m.       12p.m.    m.       12p.m.     m.        12p.m. 


FIG.  50. — Variations  in  radial  or  transverse  diameter  of  a  tomato  during  development  in  28  days. 
Increase  is  denoted  by  downward  course  of  the  auxographic  tracing,  and  the  direct  effects  of 
temperatures  and  relative  humidity  are  illustrated  by  the  record  and  the  accompanying 
notations  in  the  figure.  Amplified  5  times  on  scale  ruled  to  5  mm.  intervals. 


Imbibition  and  Growth  in  Fruits. 


169 


If  this  method  be  followed  it  would  at  once  be  obvious  that  while  the 
rate  of  increase  in  diameter  would  be  a  direct  measurement,  yet  as  the 
fruit  increases  as  a  globe  the  actual  material  added  could  be  regarded 
as  a  shell  on  this  globe.  The  rate  in  terms  of  volume  would  therefore 
be  the  amount  of  this  shell  to  be  calculated  by  finding  the  difference 
between  the  initial  volume  and  the  volume  at  the  end  of  each  period. 
The  rate  by  direct  measurement  of  diameter  and  by  volume  increases 
may  be  compared  as  hi  table  121,  for  periods  of  one  week  beginning 
on  the  date  given. 

TABLE  121. — Average  daily  rate  of  growth.  TABLE  122. 


Date. 

Diameter, 
millimeters. 

Volume, 
cubic 
millimeters. 

Aug.     9. 

1.7 

2,604 

16. 

1.1 

2,513 

21. 

0.7 

2,064 

28. 

0.4 

1,373 

Sept.    4  . 

0.28 

976 

11. 

0.17 

695 

Date. 

Diameter, 
millimeters. 

Volume, 
cubic 
millimeters. 

Aug.     9. 

0.95 

2,072 

16. 

0.7 

1,852 

21. 

0.56 

1,800 

28. 

0.3 

660 

Sept.    4. 

0.2 

508 

11. 

0.2 

560 

The  rate  on  September  11  by  direct  measurement  would  appear  to 
be  one-tenth  that  of  a  month  earlier,  yet  actually  water  and  new  mate- 
rial was  being  added  at  a  rate  equivalent  to  one-fourth  of  the  earlier 
rate.  The  radial  proportions  would  make  the  rate  on  August  21  not 
much  more  than  40  per  cent  of  the  rate  on  August  9,  while  the  increase 
in  volume  was  over  96  per  cent.  The  rate  hi  the  week  beginning 
August  28  would  appear  to  be  less  than  a  fourth  that  by  direct  measure- 
ment on  August  9,  yet  actually  the  increment  of  water  and  material  is 
more  than  half  that  in  the  younger  stage  and  smaller  size. 

A  second  plant  with  the  auxograph  arranged  to  take  axial  varia- 
tions in  the  fruits  which  measured  33  mm.  was  arranged  to  run  con- 
currently with  No.  1  and  under  identical  temperature  and  conditions 
of  moisture.  The  daily  rates  of  increase  in  diameter  were  as  shown 
in  table  122  for  weeks  beginning  on  the  dates  given. 

Here  again  the  actual  course  of  growth  as  calculated  in  terms  of 
volume  shows  that  simple  measurements  of  the  thickness  do  not  express 
the  real  values  in  growth  of  such  organs. 

The  third  test  was  made  on  a  fruit  taken  at  a  much  earlier  stage  at  a 
diameter  of  16  mm.  with  a  transverse  or  radial  bearing,  the  tempera- 
ture and  moisture  conditions  being  similar  to  those  of  1  and  2.  The 
daily  rate  of  increase  was  as  shown  in  table  123  for  the  weeks  begin- 
ning on  the  given  dates. 

The  actual  volume  of  this  fruit  at  the  close  of  the  experiment  was 
approximately  2,900  c.  mm.  and  its  growth  had  been  followed  for  a 
period  of  40  days.  It  is  notable  that  in  the  earlier  stage  in  the  advance 
of  the  fruit  from  20  to  26  mm.  in  diameter  (August  21  to  August  31), 
while  the  increase  of  the  diameter  seems  constant,  yet  the  actual 


170 


Hydration  and  Growth. 


accession  of  material  is  very  much  greater.  Then,  in  further  develop- 
ment, the  average  increment  to  the  diameter  was  smaller,  yet  the  actual 
accession  of  material  was  greater  (see  September  4).  Following  this, 
the  rate  falling  from  0.8  to  0.3  mm.  daily,  the  accession  decreases  less 
than  half.  (See  figs.  51  and  52.) 

TABLE  124. 


TABLE  123. 


Date. 

Diameter, 
millimeters. 

Volume, 
cubic 
millimeters. 

Aug.   21. 

0.85 

537 

28. 

0.85 

851 

Sept.    4. 

0.64 

885 

11. 

0.8 

1,643 

18. 

0.3 

594 

25. 

0.37 

662 

20° 

C. 

30° 

C. 

Diam. 

Volume. 

Diam. 

Volume. 

Sept.  1. 

mm. 
1 

c.  mm. 
72 

mm. 

c.  mm. 

Sept.  7.  ... 
Sept.  14.  ... 
Sept.  21.... 

2 
1.4 
0.08 

33 
33 
9 

0.8 
.3 

.085 

128 
91 
27 

Attention  was  now  directed  to  temperature  effects  as  measured  in 
this  manner.  Two  plants  were  placed  in  chambers  subjected  to  equiv- 
alent diffuse  illumination  and  humidity.  The  fruits  similar  to  those 


\ 


i 1 1 • . , 

FIG.  51. — Diagram  illustrating  the  course 
of  growth  of  a  tomato  during  the  six 
weeks  of  its  development.  The  broken 
line  is  plotted  from  the  average  daily 
rate  of  growth  during  each  week,  and 
the  solid  line  from  the  calculated  in- 
creases in  volume. 


Fro.  52. — Similar  to  fig.  51,  but  begin- 
ning at  an  earlier  stage.  The  average 
daily  rate  is  seen  to  form  a  graph  which 
presents  notable  differences  from  the 
one  plotted  from  variations  in  volume. 


measured  in  one  showed  thermometer  readings  of  19°  to  21°  C.  and 
in  the  other  29°  to  31°  C.  The  daily  rates  of  axial  increase  were  as 
shown  in  table  124  for  the  weeks  beginning  on  the  given  dates. 

The  conditions  under  which  both  plants  were  grown  were  unfavor- 
able to  development,  but  it  is  to  be  noted  that  the  rates  of  increase 


Imbibition  and  Growth  in  Fruits.  171 

sustained  a  changing  relation  as  growth  slackened.  The  enlargement 
of  such  highly  watery  fruits  must  be  so  largely  a  matter  of  diffusion 
and  hydration  that  any  formula  expressive  of  the  temperature  re- 
lations of  chemical  transformation  must  be  wide  of  the  facts  in  many 
stages  of  development. 

The  record  of  growth  of  No.  3,  which  is  given  in  full  in  figure  50, 
shows  beyond  question  the  effect  of  transpiration  and  water-loss  on 
growth.  As  the  daily  temperatures  of  the  fruits  rose  from  12°  C.  and 
14°  C.  to  26°  C.  and  28°  C.,  acceleration  ensued  up  to  a  point  where 
the  rise  caused  a  water-loss  overbalancing  the  gain  by  hydration. 
Higher  temperatures,  therefore,  did  not  facilitate  or  accelerate  growth 
unless  accompanied  by  high  relative  humidity.  Thus  the  highest 
growth  rates  are  those  of  midday  and  afternoon,  with  fog  or  showers. 
This  is  especially  marked  on  the  records  of  September  10,  11, 12,  and 
13,  in  which  a  50-hour  rainy  period  was  anticipated  and  followed  by 
high  humidity.  (See  fig.  50.)  It  was  not  possible  to  increase  the 
water-supply  by  watering  the  soil  around  the  roots  in  such  manner  as 
to  cancel  the  midday  shrinkage  or  slackening  in  growth.  One  espe- 
cially striking  effect  is  that  in  which  the  rise  in  temperature  conse- 
quent upon  the  cessation  of  the  rain,  from  20°  to  25°  C.  at  3  p.  m.  on 
September  13,  was  followed  by  a  lessened  rate  of  growth.  On  the 
cloudy  days  growth  was  uniformly  high.  Similar  effects  were  exhib- 
ited by  a  small  fruit  of  a  potato  in  a  greenhouse  at  Tucson  in  May 
1918. 

The  two  types  of  fruits  are  seen  to  show  a  concordant  behavior  with 
respect  to  the  balance  between  the  water-supply  and  transpiration. 
A  rise  in  temperature  with  accompanying  lessened  relative  humidity 
had  the  effect  of  retarding  or  stopping  growth  or  of  producing  an  actual 
shrinkage  in  volume.  The  nut  and  the  berry  are  both  more  highly 
hydrated  or  more  watery  than  the  stem  through  which  their  water- 
supply  must  be  drawn.  This  was  established  by  measurement  in  the 
walnut  and  is  obvious  with  respect  to  the  tomato  and  its  stems. 

A  distinction  must  be  made  between  the  water-relations  of  a  fruit 
and  its  stem  and  that  which  prevails  between  a  parasite  and  its  host, 
or  between  a  swelling  colloid  and  the  solution  in  which  it  may  be  im- 
mersed. The  water  deficit  of  the  stems  as  measured  by  swelling  in- 
cludes that  of  the  entire  structure.  The  fruits,  however,  receive  their 
supply  through  special  conduits  which  sustain  only  a  mechanical  rela- 
tion to  the  other  parts  of  the  stem  which  may  be  active  in  its  swelling. 
Such  non-cctaducting  tissues  of  course  draw  their  supply  from  this 
system  of  conduits  also,  but  it  is  highly  probable  that  the  dispropor- 
tion between  the  water-content  of  the  fruit  and  of  the  tracts  in  the 
stem  from  which  it  receives  its  supply  is  not  so  great  as  might  be 
indicated  by  the  measurements  given.  The  hydration  capacity  of  the 
fruits  would  be  the  resultant  of  many  factors,  including  the  pentosan- 


172  Hydration  and  Growth. 

protein  ratio,  the  hydrogen-ion  concentration,  the  action  of  salts,  and 
the  effect  of  the  amino  compounds. 

The  delicate  balance  between  water-loss  and  absorption  as  revealed 
by  measurements  of  growing  organs  of  all  kinds  is  very  striking.  The 
rate  at  which  water  is  received  is  generally  so  little  hi  excess  of  the 
transpiration  that  a  rise  of  10  to  15  degrees  centigrade  may  extinguish 
the  balance.  At  the  same  time,  such  rise  in  temperature  may  also 
result  in  a  lessened  hydration  capacity,  so  that  by  the  action  of  the 
acids  at  the  higher  temperature,  water  may  theoretically  be  forced  out 
of  the  colloidal  complex. 

It  is  plainly  evident  that  growth  consists  of  two  fundamental 
features — hydration  of  the  colloidal  material  of  the  plasma  and  the 
arrangement  of  additional  material  hi  colloidal  structures  with  the 
entailed  additional  capacity  for  adsorbing  water.  The  first  may 
occur  without  the  second,  and  increase  in  volume  might  occur 
in  a  pentosan-protein  colloid  at  any  time  by  the  action  of  its 
own  metabolic  products,  such  as  the  hydrogen-ion  concentration 
or  the  proportion  of  ammo-compounds  formed.  Growth  by  acces- 
sion of  solid  material  without  a  corresponding  absorption  of  water 
is  characteristic  of  cell  organs  or  walls,  and  such  deposition  of 
material  can  only  result  in  changes  hi  volume  which  would  not  be 
measurable  by  auxographic  methods. 

Hydration  consists,  in  the  first  instance,  of  the  union  of  molecules  of 
water  with  the  molecules  of  solid  material  in  the  colloidal  masses,  and 
it  is  this  action  which  is  entailed  in  the  initial  and  almost  instantaneous 
enlargement  of  dried  sections  when  water  is  poured  on  them.  No  serious 
reason  has  yet  been  advanced,  however,  against  the  extension  of  the 
term  to  apply  to  the  accompanying  and  subsequent  adsorption  of  an 
indefinite  number  of  molecules  on  the  surfaces  of  the  molecular  aggre- 
gates. Cell-masses  are  already  in  an  advanced  stage  of  hydration,  and 
all  of  the  tests  with  living  material  are  simply  modifications  of  such  a 
condition.  The  swelling  of  dried  sections  of  plant  tissue  may  include 
some  chemical  action,  or  some  union  of  water  with  the  solid  material 
in  definite  proportions. 

The  manner  in  which  hydration  ensues,  or  rather  the  character  of  the 
process,  will  naturally  depend  upon  the  character  of  the  cell  colloids. 
If  these  are  albuminous,  swelling  will  be  largely  determined  by 
the  hydrogen-ion  concentration  of  the  solution.  It  also  follows  that 
any  cell-organ  or  cell-mass  which  is  dominantly  proteinaceous  will 
show  such  increases  of  hydration  capacity  with  acidity,  modified  by 
other  facts,  including  the  presence  of  salts  or  bases. 

These  effects  are  modified  or  reversed  in  colloidal  material  which 
consists  more  largely  of  carbohydrate  material.  The  pentosans 
represented  by  various  gums  and  mucilages  are  abundant  in  plant 
cells,  and  these  present  some  variety  of  composition  and  differences 


Imbibition  and  Growth  in  Fruits.  173 

in  solubility  or  dispersibility.  One  group  which  may.  be  illustrated 
by  agar  has  a  definitely  limited  swelling  capacity  under  temperatures 
below  50°  C.  and  other  conditions,  and  of  course  is  not  soluble.  Others, 
like  the  mucilages  of  Opuntia  or  acacia  or  tragacanth,  are  soluble,  and 
when  placed  in  water  pass  from  a  dry  solid  state  to  a  complete  solution. 
The  solubility  of  protoplasm  will  depend  upon  the  presence  of  these 
substances,  as  well  as  upon  the  albumins  which  may  be  present. 

The  ideal  capacity  for  hydration  and  growth  of  any  mass  of  proto- 
plasm would  be  a  resultant  of  the  composition  and  proportions  of  its 
organic  material  and  of  the  relation  of  the  phases  in  which  they  occur. 
The  theoretical  maximum  hydration  of  a  carbohydrate-protein  system 
is  invariably  modified  by  the  nutrient  salts  adsorbed  in  its  structure  and 
by  the  products  of  unceasing  metabolic  changes,  especially  the  trans- 
formations which  are  comprehended  in  respiration  and  which  carry 
compounds  through  a  stage  in  which  acids  are  formed.  These  features, 
as  influenced  by  temperature,  determine  the  rate,  daily  course,  and  total 
expansion  in  growth.  In  addition,  a  certain  amount  of  material  is  lost 
from  the  plant  in  the  form  of  carbon  dioxid,  and,  as  has  been  emphasized 
on  the  preceding  pages,  the  surface  loss  of  water  may  on  occasion  be 
greater  than  the  amount  passing  into  the  growing  cell-masses.  The 
above-mentioned  processes  and  agencies  affect  the  rate,  course,  and 
amount  of  growth. 


LITERATURE  CITED. 


ASKENASY,  E.    Ueber  die  Temperature,  welche  Pflanzen  im  Sonnenlicht  annehmen.     Bot. 

Zeitung.,  33:441-2.     1875. 
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SCHOOL  U8RW 


